register interest

Professor Paul Klenerman

Research Area: Immunology
Technology Exchange: Cell sorting
Scientific Themes: Immunology & Infectious Disease
Keywords: Hepatitis C virus, HIV, T cells, virus, liver and flow cytometry
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Infectious diseases such as HIV, hepatitis B and C, affect hundreds of millions of people worldwide. Our group works on the immune response to these infections, focusing on both the host and the pathogen. As there is currently no vaccine to prevent hepatitis C infection, many of our projects have addressed aspects of chronic hepatitis C virus (HCV) infection and vaccine responses. In addition, our group also looks at a range of viruses and bacteria. Overall our main contributions to date have been to define mechanisms of viral persistence, including: T cell escape, antagonism, original antigen sin, integration of non-retroviral RNA viruses; to define the key features of successful immune responses against HCV, leading to trials of a T cell vaccine; and to define the distinctive CD161+ T cell population, which dominates in the human liver.

We are currently working on three main strands of research:

1. CD161++/MAIT cell biology. These lymphocytes, which are abundant in human blood and highly enriched in the liver, have the capacity to respond to both bacterial and inflammatory signals. We are trying to understand their in vivo role in host defence and immunopathology, through the analysis of patients and in vitro studies of function and activation.

2. HCV immune defence. In collaboration with the groups of Ellie Barnes and Adrian Hill, we are involved in vaccine studies using adenoviral vectors to track vaccine-induced cells and analyse their capacity to recognise viral variants. Upcoming studies include an analysis of host responses in relation to novel drug therapies and the use of new viral sequencing approaches to define the impact of host immunity.

3. Memory inflation. Some persistent virus infections induce a striking host response, which we have termed memory "inflation"; this includes the generation of very large functional T cell populations, which can increase with time. While this was first noted in cytomegalovirus infections, we have found a similar profile of cells after adenoviral vector vaccination. Our group is working to define the qualities of these induced cells, and the critical factors that drive memory inflation. 

Name Department Institution Country
Dr Georg Lauer Gastroenterology Harvard University United States
Robert Thimme University Hospital Freiburg Germany
Professor Mala Maini UCL United Kingdom
Professor Jonathan Flint Wellcome Trust Centre for Human Genetics University of Oxford United Kingdom
Professor Ellie (Eleanor) Barnes Experimental Medicine Division Oxford University, Peter Medawar Building United Kingdom
Professor Chris A O'Callaghan Centre for Cellular and Molecular Physiology Oxford University, Henry Wellcome Building for Molecular Physiology United Kingdom
Dr Nick Haining Harvard University United States
Ray T Chung Harvard University United States
John Wherry U Penn United States
Silvana Gaudieri University of Western Australia Australia
Andrea Cox Johns Hopkins, Baltimore United States
Ramon Arens University of Leiden Netherlands
Professor Constantino Lopez-Macias IMSS Mexico
James McCluskey University of Melbourne Australia
James Ussher university of otago New Zealand
Dr Burkhard Ludewig Kantonsspital St Gallen Switzerland
Turtle L, Bali T, Buxton G, Chib S, Chan S, Soni M, Hussain M, Isenman H et al. 2016. Human T cell responses to Japanese encephalitis virus in health and disease. J Exp Med, 213 (7), pp. 1331-1352. | Show Abstract | Read more

Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8(+) and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4(+) and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4(+) T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4(+) and CD8(+) T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus.

Klenerman P, Oxenius A. 2016. T cell responses to cytomegalovirus. Nat Rev Immunol, 16 (6), pp. 367-377. | Show Abstract | Read more

Human cytomegalovirus (HCMV) establishes a latent infection that generally remains asymptomatic in immune-competent hosts for decades but can cause serious illness in immune-compromised individuals. The long-term control of CMV requires considerable effort from the host immune system and has a lasting impact on the profile of the immune system. One hallmark of CMV infection is the maintenance of large populations of CMV-specific memory CD8(+) T cells - a phenomenon termed memory inflation - and emerging data suggest that memory inflation is associated with impaired immunity in the elderly. In this Review, we discuss the molecular triggers that promote memory inflation, the idea that memory inflation could be considered a natural pathway of T cell maturation that could be harnessed in vaccination, and the broader implications of CMV infection and the T cell responses it elicits.

Ussher JE, van Wilgenburg B, Hannaway RF, Ruustal K, Phalora P, Kurioka A, Hansen TH, Willberg CB, Phillips RE, Klenerman P. 2016. TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells. Eur J Immunol, 46 (7), pp. 1600-1614. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1-mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1-mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1-mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B-cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B-cell lines, suggesting differential regulation in different cell types. APC activation and NF-κB signaling were critical for MR1-mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1-mediated MAIT cell activation. Overall, MR1-mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.

Culver EL, Sadler R, Simpson D, Cargill T, Makuch M, Bateman AC, Ellis AJ, Collier J et al. 2016. Elevated Serum IgG4 Levels in Diagnosis, Treatment Response, Organ Involvement, and Relapse in a Prospective IgG4-Related Disease UK Cohort. Am J Gastroenterol, 111 (5), pp. 733-743. | Show Abstract | Read more

OBJECTIVES: Elevated serum immunoglobulin G4 (IgG4) levels have been associated with autoimmune pancreatitis and IgG4-related disease (IgG4-RD) for over a decade. However, an elevated serum IgG4 is not specific for the disease. There have been inconsistent reports of its use in diagnosis, as a marker of disease relapse, and its relationship to organ involvement in retrospective cohorts. The aims of this study were to ascertain conditions that are associated with an elevated serum IgG4 and to investigate the role of IgG4 in diagnosis, relapse, and organ involvement in a prospective cohort of patients with IgG4-RD. METHODS: We evaluated serum IgG4 measurements in the Oxford Immunology Laboratory over 6 years. Patients in whom serum IgG4 was requested to differentiate IgG4-RD from other diseases were recruited into a longitudinal follow-up study to determine final diagnosis. In a prospective cohort of IgG4-RD patients, organ involvement, response to therapy, and disease relapse were determined. RESULTS: Two thousand and sixty-seven samples from 1,510 patients had serum IgG4 measured. Of these, IgG4 was elevated (≥1.4 g l(-1)) in 243 (16.1%) patients. The main indication (85.6%) was to distinguish between IgG4-RD and non-IgG4-RD conditions. Only 5.1% of patients who had serum IgG4 measured for this purpose had a final diagnosis of IgG4-RD. Of those with an elevated serum IgG4, 22.4% met IgG4-RD diagnostic criteria. Serum IgG4 was elevated in 48 (82.8%) of IgG4-RD patients. An IgG4 cutoff of 1.4 g l(-1) gave a sensitivity of 82.8% and specificity of 84.7% to diagnose IgG4-RD. Increasing this to 2.8 g l(-1) increased specificity to 96.2% and negative predictive value to 97.7%, with a lower sensitivity of 56.9% and positive predictive value of 44.5%. Serum IgG4 levels fell with corticosteroid therapy, but this was not disease-specific. A serum IgG4 of ≥2.8 g l(-1) at diagnosis was associated with multi-organ involvement and risk of relapse. CONCLUSIONS: Serum IgG4 levels are elevated in multiple non-IgG4-RD inflammatory and malignant conditions, with less than one-quarter of those with an elevated IgG4 meeting IgG4-RD diagnostic criteria. A serum IgG4 of ≥2.8 g l(-1) is useful in distinguishing between IgG4-RD and non-IgG4-RD diagnoses, predicting multiple-organ involvement and risk of relapse in IgG4-RD.

Bonsall D, Gregory WF, Ip CL, Donfield S, Iles J, Ansari MA, Piazza P, Trebes A et al. 2016. Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR. Emerg Infect Dis, 22 (4), pp. 671-678. | Show Abstract | Read more

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.

Sylwester A, Nambiar KZ, Caserta S, Klenerman P, Picker LJ, Kern F. 2016. A new perspective of the structural complexity of HCMV-specific T-cell responses. Mech Ageing Dev, 158 pp. 14-22. | Show Abstract | Read more

BACKGROUND: In studies exploring the effects of HCMV infection on immune system aging ('immunosenescence'), after organ transplantation or in other settings, HCMV-specific T-cell responses are often assessed with respect to purportedly 'immunodominant' protein subunits. However, the response structure in terms of recognized antigens and response hierarchies (architecture) is not well understood and actual correlates of immune protection are not known. METHODS: We explored the distribution of T-cell response sizes and dominance hierarchies as well as response breadth in 33 HCMV responders with respect to >200 HCMV proteins. RESULTS: At the individual responder level HCMV-specific T-cell responses were generally arranged in clear dominance hierarchies; interestingly, the number of proteins recognized by an individual correlated closely with the size of their biggest response. Target-specificity varied considerably between donors and across hierarchy levels with the presence, size, and hierarchy position of responses to purportedly 'immunodominant' targets being unpredictable. CONCLUSIONS: Predicting protective immunity based on isolated HCMV subunit-specific T-cell responses is questionable in light of the complex architecture of the overall response. Our findings have important implications for T-cell monitoring, intervention strategies, as well as the application of animal models to the understanding of human infection.

Colston JM, Bolinger B, Cottingham MG, Gilbert S, Klenerman P. 2016. Modification of Antigen Impacts on Memory Quality after Adenovirus Vaccination. J Immunol, 196 (8), pp. 3354-3363. | Show Abstract | Read more

The establishment of robust T cell memory is critical for the development of novel vaccines for infections and cancers. Classical memory generated by CD8(+)T cells is characterized by contracted populations homing to lymphoid organs. T cell memory inflation, as seen for example after CMV infection, is the maintenance of expanded, functional, tissue-associated effector memory cell pools. Such memory pools may also be induced after adenovirus vaccination, and we recently defined common transcriptional and phenotypic features of these populations in mice and humans. However, the rules that govern which epitopes drive memory inflation compared with classical memory are not fully defined, and thus it is not currently possible to direct this process. We used our adenoviral model of memory inflation to first investigate the role of the promoter and then the role of the epitope context in determining memory formation. Specifically, we tested the hypothesis that conventional memory could be converted to inflationary memory by simple presentation of the Ag in the form of minigene vectors. When epitopes from LacZ and murine CMV that normally induce classical memory responses were presented as minigenes, they induced clear memory inflation. These data demonstrate that, regardless of the transgene promoter, the polypeptide context of a CD8(+)T cell epitope may determine whether classical or inflating memory responses are induced. The ability to direct this process by the use of minigenes is relevant to the design of vaccines and understanding of immune responses to pathogens.

Pedergnana V, Smith D, STOP-HCV Consortium, Klenerman P, Barnes E, Spencer CC, Ansari MA. 2016. Interferon Lambda 4 variant rs12979860 is not associated with RAV NS5A Y93H in Hepatitis C Virus Genotype 3a. Hepatology, pp. n/a-n/a. | Read more

Fergusson JR, Hühn MH, Swadling L, Walker LJ, Kurioka A, Llibre A, Bertoletti A, Holländer G et al. 2016. CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut. Mucosal Immunol, 9 (2), pp. 401-413. | Show Abstract | Read more

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Gaunt E, Wise HM, Zhang H, Lee LN, Atkinson NJ, Nicol MQ, Highton AJ, Klenerman P et al. 2016. Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection. Elife, 5 (FEBRUARY2016), pp. e12735. | Show Abstract | Read more

Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.

Jeffery HC, van Wilgenburg B, Kurioka A, Parekh K, Stirling K, Roberts S, Dutton EE, Hunter S et al. 2016. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1. J Hepatol, 64 (5), pp. 1118-1127. | Show Abstract | Read more

BACKGROUND & AIMS: Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. METHODS: The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. RESULTS: Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. CONCLUSIONS: Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future.

Arriaga-Pizano L, Ferat-Osorio E, Rodríguez-Abrego G, Mancilla-Herrera I, Domínguez-Cerezo E, Valero-Pacheco N, Pérez-Toledo M, Lozano-Patiño F et al. 2015. Differential Immune Profiles in Two Pandemic Influenza A(H1N1)pdm09 Virus Waves at Pandemic Epicenter. Arch Med Res, 46 (8), pp. 651-658. | Show Abstract | Read more

BACKGROUND AND AIMS: Severe influenza A(H1N1)pdm2009 virus infection cases are characterized by sustained immune activation during influenza pandemics. Seasonal flu data suggest that immune mediators could be modified by wave-related changes. Our aim was to determine the behavior of soluble and cell-related mediators in two waves at the epicenter of the 2009 influenza pandemic. METHODS: Leukocyte surface activation markers were studied in serum from peripheral blood samples, collected from the 1(st) (April-May, 2009) and 2(nd) (October 2009-February 2010) pandemic waves. Patients with confirmed influenza A(H1N1)pdm2009 virus infection (H1N1), influenza-like illness (ILI) or healthy donors (H) were analyzed. RESULTS: Serum IL-6, IL-4 and IL-10 levels were elevated in H1N1 patients from the 2(nd) pandemic wave. Additionally, the frequency of helper and cytotoxic T cells was reduced during the 1(st) wave, whereas CD69 expression in helper T cells was increased in the 2(nd) wave for both H1N1 and ILI patients. In contrast, CD62L expression in granulocytes from the ILI group was increased in both waves but in monocytes only in the 2(nd) wave. Triggering Receptor Expressed on Myeloid cells (TREM)-1 expression was elevated only in H1N1 patients at the 1(st) wave. CONCLUSIONS: Our results show that during the 2009 influenza pandemic a T cell activation phenotype is observed in a wave-dependent fashion, with an expanded activation in the 2(nd) wave, compared to the 1(st) wave. Conversely, granulocyte and monocyte activation is infection-dependent. This evidence collected at the pandemic epicenter in 2009 could help us understand the differences in the underlying cellular mechanisms that drive the wave-related immune profile behaviors that occur against influenza viruses during pandemics.

Matthews PC, Carlson JM, Beloukas A, Malik A, Jooste P, Ogwu A, Shapiro R, Riddell L et al. 2016. HLA-A is a Predictor of Hepatitis B e Antigen Status in HIV-Positive African Adults. J Infect Dis, 213 (8), pp. 1248-1252. | Show Abstract | Read more

Outcomes of chronic infection with hepatitis B virus (HBV) are varied, with increased morbidity reported in the context of human immunodeficiency virus (HIV) coinfection. The factors driving different outcomes are not well understood, but there is increasing interest in an HLA class I effect. We therefore studied the influence of HLA class I on HBV in an African HIV-positive cohort. We demonstrated that virologic markers of HBV disease activity (hepatitis B e antigen status or HBV DNA level) are associated with HLA-A genotype. This finding supports the role of the CD8(+) T-cell response in HBV control, and potentially informs future therapeutic T-cell vaccine strategies.

Matthews P, Beloukas A, Malik A, Carlson J, Jooste P, Ogwu A, Shapiro R, Riddell L et al. 2015. HLA CONTRIBUTES TO IMMUNE CONTROL OF HEPATITIS B IN HIV-POSITIVE AFRICAN ADULTS JOURNAL OF INFECTION, 71 (6), pp. 685-686. | Read more

Klenerman P, Fitzmaurice K. 2015. An update on hepatitis C virus. Clin Med (Lond), 15 Suppl 6 (Suppl_6), pp. s33-s36. | Show Abstract | Read more

Chronic hepatitis caused by hepatitis C virus infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma globally. Over the last few years, there have been significant advances in our understanding of the immunopathogenesis of this infection, as well as revolutionary developments in the therapeutic armamentarium. The introduction of a number of new oral antiviral agents is already delivering major benefits to patients as they are generally well tolerated and are associated with excellent response rates in the majority. Nonetheless, a number of clinical scenarios remain challenging and research is ongoing in these areas. In this update, we review developments in both basic as well as translational research in the field.

Bolinger B, Sims S, Swadling L, O'Hara G, de Lara C, Baban D, Saghal N, Lee LN et al. 2015. Adenoviral Vector Vaccination Induces a Conserved Program of CD8(+) T Cell Memory Differentiation in Mouse and Man. Cell Rep, 13 (8), pp. 1578-1588. | Show Abstract | Read more

Following exposure to vaccines, antigen-specific CD8(+) T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8(+) T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8(+) T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8(+) T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses.

Klenerman P, Smith T, Horan F, Ross AC. 2015. Professor Leslie Klenerman (1929 to 2015). Bone Joint J, 97-B (11), pp. 1588. | Read more

Kelly C, Swadling L, Capone S, Brown A, Richardson R, Halliday J, von Delft A, Oo Y et al. 2016. Chronic hepatitis C viral infection subverts vaccine-induced T-cell immunity in humans. Hepatology, 63 (5), pp. 1455-1470. | Show Abstract | Read more

UNLABELLED: Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high-magnitude, durable CD4(+) and CD8(+) T-cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV-specific immune responses and determine T-cell cross-reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1-infected patients were vaccinated using heterologous adenoviral vectors (ChAd3-NSmut and Ad6-NSmut) encoding HCV NS proteins in a dose escalation, prime-boost regimen, with and without concomitant pegylated interferon-α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon-γ enzyme-linked immunospot assays. Cross-reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV-specific T-cell responses following vaccination was markedly reduced. CD8(+) HCV-specific T-cell responses were detected in 15/24 patients at the highest dose, whereas CD4(+) T-cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T-cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. CONCLUSION: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T-cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections.

Adland E, Klenerman P, Goulder P, Matthews PC. 2015. Ongoing burden of disease and mortality from HIV/CMV coinfection in Africa in the antiretroviral therapy era. Front Microbiol, 6 (SEP), pp. 1016. | Show Abstract | Read more

Human Cytomegalovirus (CMV) is a well-recognized pathogen in the context of HIV infection, but since the roll out of ART, clinical and scientific interest in the problem of HIV/CMV coinfection has diminished. However, CMV remains a significant cofactor in HIV disease, with an influence on HIV acquisition, disease progression, morbidity, and mortality. Disease manifestations may be a result of direct interplay between the two viruses, or may arise as a secondary consequence of immune dysregulation and systemic inflammation. The problem is most relevant when the rates of coinfection are high, most notably in sub-Saharan Africa, and in children at risk of acquiring both infections early in life. Understanding the interplay between these viruses and developing strategies to diagnose, treat and prevent CMV should be a priority.

Wikramaratna PS, Lourenço J, Klenerman P, Pybus OG, Gupta S. 2015. Effects of neutralizing antibodies on escape from CD8+ T-cell responses in HIV-1 infection. Philos Trans R Soc Lond B Biol Sci, 370 (1675), pp. 20140290-20140290. | Show Abstract | Read more

Despite substantial advances in our knowledge of immune responses against HIV-1 and of its evolution within the host, it remains unclear why control of the virus eventually breaks down. Here, we present a new theoretical framework for the infection dynamics of HIV-1 that combines antibody and CD8(+) T-cell responses, notably taking into account their different lifespans. Several apparent paradoxes in HIV pathogenesis and genetics of host susceptibility can be reconciled within this framework by assigning a crucial role to antibody responses in the control of viraemia. We argue that, although escape from or progressive loss of quality of CD8(+) T-cell responses can accelerate disease progression, the underlying cause of the breakdown of virus control is the loss of antibody induction due to depletion of CD4(+) T cells. Furthermore, strong antibody responses can prevent CD8(+) T-cell escape from occurring for an extended period, even in the presence of highly efficacious CD8(+) T-cell responses.

Green CA, Scarselli E, Sande CJ, Thompson AJ, de Lara CM, Taylor KS, Haworth K, Del Sorbo M et al. 2015. Chimpanzee adenovirus- and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults. Sci Transl Med, 7 (300), pp. 300ra126. | Show Abstract | Read more

Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease.

Iles JC, Njouom R, Foupouapouognigni Y, Bonsall D, Bowden R, Trebes A, Piazza P, Barnes E, Pépin J, Klenerman P, Pybus OG. 2015. Characterization of Hepatitis C Virus Recombination in Cameroon by Use of Nonspecific Next-Generation Sequencing. J Clin Microbiol, 53 (10), pp. 3155-3164. | Show Abstract | Read more

The importance of recombination in the evolution and genetic diversity of the hepatitis C virus (HCV) is currently uncertain. Only a small number of intergenotypic recombinants have been identified so far, and each has core and envelope genes classified as belonging to genotype 2. Here, we investigated two putative genotype 4/1 recombinants from southern Cameroon using a number of approaches, including standard Sanger sequencing, genotype-specific PCR amplification, and non-HCV-specific Illumina RNA sequencing (RNA-seq). Recombination between genotypes 1 and 4 was confirmed in both samples, and the parental lineages of each recombinant belong to HCV subtypes that are cocirculating at a high prevalence in Cameroon. Using the RNA-seq approach, we obtained a complete genome for one sample, which contained a recombination breakpoint at the E2/P7 gene junction. We developed and applied a new method, called Deep SimPlot, which can be used to visualize and identify viral recombination directly from the short sequence reads created by next-generation sequencing in conjunction with a consensus sequence.

van Gool IC, Eggink FA, Freeman-Mills L, Stelloo E, Marchi E, de Bruyn M, Palles C, Nout RA et al. 2015. POLE Proofreading Mutations Elicit an Antitumor Immune Response in Endometrial Cancer. Clin Cancer Res, 21 (14), pp. 3347-3355. | Show Abstract | Read more

PURPOSE: Recent studies have shown that 7% to 12% of endometrial cancers are ultramutated due to somatic mutation in the proofreading exonuclease domain of the DNA replicase POLE. Interestingly, these tumors have an excellent prognosis. In view of the emerging data linking mutation burden, immune response, and clinical outcome in cancer, we investigated whether POLE-mutant endometrial cancers showed evidence of increased immunogenicity. EXPERIMENTAL DESIGN: We examined immune infiltration and activation according to tumor POLE proofreading mutation in a molecularly defined endometrial cancer cohort including 47 POLE-mutant tumors. We sought to confirm our results by analysis of RNAseq data from the TCGA endometrial cancer series and used the same series to examine whether differences in immune infiltration could be explained by an enrichment of immunogenic neoepitopes in POLE-mutant endometrial cancers. RESULTS: Compared with other endometrial cancers, POLE mutants displayed an enhanced cytotoxic T-cell response, evidenced by increased numbers of CD8(+) tumor-infiltrating lymphocytes and CD8A expression, enrichment for a tumor-infiltrating T-cell gene signature, and strong upregulation of the T-cell cytotoxic differentiation and effector markers T-bet, Eomes, IFNG, PRF, and granzyme B. This was accompanied by upregulation of T-cell exhaustion markers, consistent with chronic antigen exposure. In silico analysis confirmed that POLE-mutant cancers are predicted to display more antigenic neoepitopes than other endometrial cancers, providing a potential explanation for our findings. CONCLUSIONS: Ultramutated POLE proofreading-mutant endometrial cancers are characterized by a robust intratumoral T-cell response, which correlates with, and may be caused by an enrichment of antigenic neopeptides. Our study provides a plausible mechanism for the excellent prognosis of these cancers.

von Delft A, Humphreys IS, Brown A, Pfafferott K, Lucas M, Klenerman P, Lauer GM, Cox AL, Gaudieri S, Barnes E. 2016. The broad assessment of HCV genotypes 1 and 3 antigenic targets reveals limited cross-reactivity with implications for vaccine design. Gut, 65 (1), pp. 112-123. | Show Abstract | Read more

OBJECTIVE: Developing a vaccine that is cross-reactive between HCV genotypes requires data on T cell antigenic targets that extends beyond genotype-1. We characterised T cell immune responses against HCV genotype-3, the most common infecting genotype in the UK and Asia, and assessed within genotype and between genotype cross-reactivity. DESIGN: T cell targets were identified in 140 subjects with either acute, chronic or spontaneously resolved HCV genotype-3 infection using (1) overlapping peptides and (2) putative human leucocyte antigens (HLA)-class-I wild type and variant epitopes through the prior assessment of polymorphic HCV genomic sites associated with host HLA, in IFNγ-ELISpot assays. CD4+/CD8+ T cell subsets were defined and viral variability at T cell targets was determined through population analysis and viral sequencing. T cell cross-reactivity between genotype-1 and genotype-3 variants was assessed. RESULTS: In resolved genotype-3 infection, T cells preferentially targeted non-structural proteins at a high magnitude, whereas in chronic disease T cells were absent or skewed to target structural proteins. Additional responses to wild type but not variant HLA predicted peptides were defined. Major sequence viral variability was observed within genotype-3 and between genotypes 1 and 3 HCV at T cell targets in resolved infection and at dominant epitopes, with limited T cell cross-reactivity between viral variants. Overall 41 CD4/CD8+ genotype-3 T cell targets were identified with minimal overlap with those described for HCV genotype-1. CONCLUSIONS: HCV T cell specificity is distinct between genotypes with limited T cell cross-reactivity in resolved and chronic disease. Therefore, viral regions targeted in natural HCV infection may not serve as attractive targets for a vaccine that aims to protect against multiple HCV genotypes.

Wang Q, Sachse P, Semmo M, Lokhande M, Montani M, Dufour JF, Zoulim F, Klenerman P, Semmo N. 2015. T- and B-cell responses and previous exposure to hepatitis B virus in 'anti-HBc alone' patients. J Viral Hepat, 22 (12), pp. 1068-1078. | Show Abstract | Read more

A serologic response to hepatitis B virus (HBV) defined as 'anti-HBc alone' is commonly observed, but its significance remains unclear. This study aimed to define the relationship between 'anti-HBc alone' serostatus and HBV infection, including HBV-specific T- and B-cell memory responses. We enrolled 31 'anti-HBc alone' patients. Total HBV DNA and cccDNA were tested by nested polymerase chain reaction (PCR) analysis in liver samples from 22 'anti-HBc alone' patients vs controls (chronic or resolved HBV infection), followed by HBsAg/HBcAg immunohistochemical (IHC) staining. IFN-γ secretion by HBV-specific T cells was compared in individuals who were 'anti-HBc alone' (n = 27), resolved HBV (n = 21), chronic HBV (n = 24) and 12 healthy controls using enzyme-linked immunospot (ELISpot) assays. An HBsAg-IgG B-cell ELISpot assay was performed in 'anti-HBc alone' patients before and after one dose of recombinant HBsAg vaccine. The majority (23/31, 74.2%) of the 'anti-HBc alone' individuals were co-infected with HCV. Infrequent intrahepatic total HBV DNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T-cell responses were similar between 'anti-HBc alone' individuals and HBV resolvers. Circulating HBV-memory B-cell responses were detected in all 'anti-HBc alone' individuals, consistent with an HBsAg-specific memory pool. After one HBV vaccine dose, increased anti-HBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P = 0.0226). 'Anti-HBc alone' individuals showed HBV-specific T-cell and memory B-cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

Hullegie SJ, Arends JE, Rijnders BJ, Irving WL, Salmon D, Prins M, Wensing AM, Klenerman P et al. 2015. Current knowledge and future perspectives on acute hepatitis C infection. Clin Microbiol Infect, 21 (8), pp. 797.e9-797.e17. | Show Abstract | Read more

Acute hepatitis C virus (HCV) infections are frequently seen worldwide in certain risk groups, with an annual incidence rate varying between 0.08% and 66%. Although this incidence is substantial, a delayed diagnosis during chronic infection is most often made in the absence of clinical symptoms in the acute phase of the infection. Currently used methods to diagnose acute HCV infection are IgG antibody seroconversion and repeated HCV RNA measurements, although no definitive diagnostic test is currently available. Progress in the field of adaptive and innate immune responses has aided both advances in the field of HCV vaccine development and a more basic understanding of viral persistence. The rapid changes in the treatment of chronic HCV infection will affect therapeutic regimens for acute HCV infection in the coming years, leading to shorter treatment courses and pegylated interferon-free modalities. This review gives an overview of the current knowledge and uncertainties, together with some future perspectives on acute hepatitis C epidemiology, virology, immunology, and treatment.

Culver EL, Vermeulen E, Makuch M, van Leeuwen A, Sadler R, Cargill T, Klenerman P, Aalberse RC, van Ham SM, Barnes E, Rispens T. 2015. Increased IgG4 responses to multiple food and animal antigens indicate a polyclonal expansion and differentiation of pre-existing B cells in IgG4-related disease. Ann Rheum Dis, 74 (5), pp. 944-947. | Show Abstract | Read more

BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition, characterised by an elevated serum IgG4 concentration and abundant IgG4-positive plasma cells in the involved organs. An important question is whether the elevated IgG4 response is causal or a reflection of immune-regulatory mechanisms of the disease. OBJECTIVES: To investigate if the IgG4 response in IgG4-RD represents a generalised polyclonal amplification by examining the response to common environmental antigens. METHODS: Serum from 24 patients with IgG4-RD (14 treatment-naive, 10 treatment-experienced), 9 patients with primary sclerosing cholangitis and an elevated serum IgG4 (PSC-high IgG4), and 18 healthy controls were tested against egg white and yolk, milk, banana, cat, peanut, rice and wheat antigens by radioimmunoassay. RESULTS: We demonstrated an elevated polyclonal IgG4 response to multiple antigens in patients with IgG4-RD and in PSC-high IgG4, compared with healthy controls. There was a strong correlation between serum IgG4 and antigen-specific responses. Responses to antigens were higher in treatment-naive compared with treatment-experienced patients with IgG4-RD. Serum electrophoresis and immunofixation demonstrated polyclonality. CONCLUSIONS: This is the first study to show enhanced levels of polyclonal IgG4 to multiple antigens in IgG4-RD. This supports that elevated IgG4 levels reflect an aberrant immunological regulation of the overall IgG4 response, but does not exclude that causality of disease could be antigen-driven.

Sims S, Bolinger B, Klenerman P. 2015. Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells (vol 45, pg, 113, 2015) EUROPEAN JOURNAL OF IMMUNOLOGY, 45 (2), pp. 636-636. | Read more

Li C, Barnes E, Newton PN, Fu Y, Vongsouvath M, Klenerman P, Okamoto H, Abe K, Pybus OG, Lu L. 2015. An expanded taxonomy of hepatitis C virus genotype 6: Characterization of 22 new full-length viral genomes. Virology, 476 pp. 355-363. | Show Abstract | Read more

We characterized the full-length genomes of 22 hepatitis C virus genotype 6 (HCV-6) isolates: 10 from Vietnam (classified into subtypes 6e, 6h, 6p, 6r, 6s, and 6u), one from China (confirmed as a new subtype 6xd), and 11 from the Lao PDR (representing a new subtype 6xe plus eight novel variants). With these 22 new genomes, HCV-6 now has a diverse and extended taxonomic structure, comprised of 28 assigned subtypes (denoted 6a-6xe) and 27 unassigned lineages, all of which have been represented by full-length genomes. Our phylogenetic analyses also included many partially-sequenced novel variants of HCV-6 from Lao PDR. This revealed that Lao HCV isolates are genetically very diverse and are phylogenetically distributed in multiple lineages within genotype 6. Our results suggest that HCV-6 has been maintained in Laos, a landlocked country, since the common ancestor of genotype 6 and indicates historical dispersal of HCV-6 across Southeast Asia.

Bonsall D, Ansari MA, Ip C, Trebes A, Brown A, Klenerman P, Buck D, STOP-HCV Consortium, Piazza P, Barnes E, Bowden R. 2015. ve-SEQ: Robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens. F1000Res, 4 pp. 1062. | Show Abstract | Read more

The routine availability of high-depth virus sequence data would allow the sensitive detection of resistance-associated variants that can jeopardize HIV or hepatitis C virus (HCV) treatment. We introduce ve-SEQ, a high-throughput method for sequence-specific enrichment and characterization of whole-virus genomes at up to 20% divergence from a reference sequence and 1,000-fold greater sensitivity than direct sequencing. The extreme genetic diversity of HCV led us to implement an algorithm for the efficient design of panels of oligonucleotide probes to capture any sequence among a defined set of targets without detectable bias. ve-SEQ enables efficient detection and sequencing of any HCV genome, including mixtures and intra-host variants, in a single experiment, with greater tolerance of sequence diversity than standard amplification methods and greater sensitivity than metagenomic sequencing, features that are directly applicable to other pathogens or arbitrary groups of target organisms, allowing the combination of sensitive detection with sequencing in many settings.

Green CA, Scarselli E, Voysey M, Capone S, Vitelli A, Nicosia A, Cortese R, Thompson AJ et al. 2015. Safety and immunogenicity of novel respiratory syncytial virus (RSV) vaccines based on the RSV viral proteins F, N and M2-1 encoded by simian adenovirus (PanAd3-RSV) and MVA (MVA-RSV); protocol for an open-label, dose-escalation, single-centre, phase 1 clinical trial in healthy adults. BMJ Open, 5 (10), pp. e008748. | Show Abstract | Read more

INTRODUCTION: Respiratory syncytial virus (RSV) infection causes respiratory disease throughout life, with infants and the elderly at risk of severe disease and death. RSV001 is a phase 1 (first-in-man), open-label, dose-escalation, clinical trial of novel genetic viral-vectored vaccine candidates PanAd3-RSV and modified vaccinia virus Ankara (MVA)-RSV. The objective of RSV001 is to characterise the (primary objective) safety and (secondary objective) immunogenicity of these vaccines in healthy younger and older adults. METHODS AND ANALYSIS: Heterologous and homologous 'prime'/boost combinations of PanAd3-RSV and single-dose MVA-RSV are evaluated in healthy adults. 40 healthy adults aged 18-50 years test one of four combinations of intramuscular (IM) or intranasal (IN) PanAd3-RSV prime and IM PanAd3 or IM MVA-RSV boost vaccination, starting at a low dose for safety. The following year an additional 30 healthy adults aged 60-75 years test either a single dose of IM MVA-RSV, one of three combinations of IN or IM PanAd3-RSV prime and PanAd3-RSV or MVA-RSV boost vaccination used in younger volunteers, and a non-vaccinated control group. Study participants are self-selected volunteers who satisfy the eligibility criteria and are assigned to study groups by sequential allocation. Safety assessment includes the daily recording of solicited and unsolicited adverse events for 1 week after vaccination, as well as visit (nursing) observations and safety bloods obtained at all scheduled attendances. Laboratory measures of RSV-specific humoral and cellular immune responses after vaccination will address the secondary end points. All study procedures are performed at the Centre for Clinical Vaccinology and Tropical Medicine (CCVTM), Oxford, UK. ETHICS AND DISSEMINATION: RSV001 has clinical trial authorisation from the Medicines and Healthcare Products Regulatory Agency (MHRA) and ethics approval from NRES Berkshire (reference 13/SC/0023). All study procedures adhere to International Conference on Harmonisation (ICH) Good Clinical Practice guidelines. The results of the trial are to be published in peer-reviewed journals, conferences and academic forums. TRIAL REGISTRATION NUMBER: NCT01805921.

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG et al. 2015. Prostaglandin D<inf>2</inf> and leukotriene E<inf>4</inf> synergize to stimulate diverse T<inf>H</inf>2 functions and T<inf>H</inf>2 cell/neutrophil crosstalk Journal of Allergy and Clinical Immunology, 135 (5), pp. 1358-1366e11. | Show Abstract | Read more

© 2015 American Academy of Allergy, Asthma & Immunology.Background Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. Objective We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. Methods The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. Results PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. Conclusions PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Gupta PK, Godec J, Wolski D, Adland E, Yates K, Pauken KE, Cosgrove C, Ledderose C et al. 2015. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells. PLoS Pathog, 11 (10), pp. e1005177. | Show Abstract | Read more

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

Wang Q, Sachse P, Semmo M, Lokhande M, Montani M, Dufour JF, Zoulim F, Klenerman P, Semmo N. 2015. T- and B-cell responses and previous exposure to hepatitis B virus in 'anti-HBc alone' patients Journal of Viral Hepatitis, 22 (12), pp. 1068-1078. | Show Abstract | Read more

© 2015 John Wiley & Sons Ltd.A serologic response to hepatitis B virus (HBV) defined as 'anti-HBc alone' is commonly observed, but its significance remains unclear. This study aimed to define the relationship between 'anti-HBc alone' serostatus and HBV infection, including HBV-specific T- and B-cell memory responses. We enrolled 31 'anti-HBc alone' patients. Total HBV DNA and cccDNA were tested by nested polymerase chain reaction (PCR) analysis in liver samples from 22 'anti-HBc alone' patients vs controls (chronic or resolved HBV infection), followed by HBsAg/HBcAg immunohistochemical (IHC) staining. IFN-γ secretion by HBV-specific T cells was compared in individuals who were 'anti-HBc alone' (n = 27), resolved HBV (n = 21), chronic HBV (n = 24) and 12 healthy controls using enzyme-linked immunospot (ELISpot) assays. An HBsAg-IgG B-cell ELISpot assay was performed in 'anti-HBc alone' patients before and after one dose of recombinant HBsAg vaccine. The majority (23/31, 74.2%) of the 'anti-HBc alone' individuals were co-infected with HCV. Infrequent intrahepatic total HBV DNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T-cell responses were similar between 'anti-HBc alone' individuals and HBV resolvers. Circulating HBV-memory B-cell responses were detected in all 'anti-HBc alone' individuals, consistent with an HBsAg-specific memory pool. After one HBV vaccine dose, increased anti-HBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P = 0.0226). 'Anti-HBc alone' individuals showed HBV-specific T-cell and memory B-cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

Hullegie SJ, Arends JE, Rijnders BJA, Irving WL, Salmon D, Prins M, Wensing AM, Klenerman P et al. 2015. Current knowledge and future perspectives on acute hepatitis C infection Clinical Microbiology and Infection, 21 (8), pp. e797.e9-797.e17. | Show Abstract | Read more

© 2015 European Society of Clinical Microbiology and Infectious Diseases.Acute hepatitis C virus (HCV) infections are frequently seen worldwide in certain risk groups, with an annual incidence rate varying between 0.08% and 66%. Although this incidence is substantial, a delayed diagnosis during chronic infection is most often made in the absence of clinical symptoms in the acute phase of the infection. Currently used methods to diagnose acute HCV infection are IgG antibody seroconversion and repeated HCV RNA measurements, although no definitive diagnostic test is currently available. Progress in the field of adaptive and innate immune responses has aided both advances in the field of HCV vaccine development and a more basic understanding of viral persistence. The rapid changes in the treatment of chronic HCV infection will affect therapeutic regimens for acute HCV infection in the coming years, leading to shorter treatment courses and pegylated interferon-free modalities. This review gives an overview of the current knowledge and uncertainties, together with some future perspectives on acute hepatitis C epidemiology, virology, immunology, and treatment.

Matthews PC, Beloukas A, Malik A, Carlson JM, Jooste P, Ogwu A, Shapiro R, Riddell L et al. 2015. Prevalence and Characteristics of Hepatitis B Virus (HBV) Coinfection among HIV-Positive Women in South Africa and Botswana. PLoS One, 10 (7), pp. e0134037. | Show Abstract | Read more

There is progressive concern about the evolving burden of morbidity and mortality caused by coinfection with HIV-1 and hepatitis B virus (HBV) in sub-Saharan Africa, but the epidemiology and impact of this problem are not well defined. We therefore set out to assimilate more information about the nature of HBV/HIV coinfection in this region by undertaking a retrospective observational study of southern African adult women. We used samples from previously recruited HIV-1 positive women attending antenatal clinics in three settings in South Africa and Botswana (n = 950) and added a small cohort of HIV-negative antenatal South African women for comparison (n = 72). We tested for HBsAg and followed up HBsAg-positive samples by testing for HBeAg, HBV DNA, HBV genotype, presence of drug-resistance associated mutations (RAMs) and HDV. We identified HBsAg in 72 individuals (7% of the whole cohort), of whom 27% were HBeAg-positive, and the majority HBV genotypes A1 and A2. We did not detect any HDV coinfection. HBV prevalence was significantly different between geographically distinct cohorts, but did not differ according to HIV status. Among adults from South Africa, HBV/HIV coinfected patients had lower CD4+ T cell counts compared to those with HIV-monoinfection (p = 0.02), but this finding was not replicated in the cohort from Botswana. Overall, these data provide a snapshot of the coinfection problem at the heart of the HIV/HBV co-epidemic, and are important to inform public health policy, resource allocation, education, surveillance and clinical care.

Matthews PC, Sharp CP, Malik A, Gregory WF, Adland E, Jooste P, Goulder PJ, Simmonds P, Klenerman P. 2015. Human parvovirus 4 infection among mothers and children in South Africa. Emerg Infect Dis, 21 (4), pp. 713-715. | Read more

Lokhande MU, Thimme R, Klenerman P, Semmo N. 2015. Methodologies for the Analysis of HCV-Specific CD4(+) T Cells. Front Immunol, 6 (FEB), pp. 57. | Show Abstract | Read more

Virus-specific CD4(+) T cells play a major role in viral infections, such as hepatitis C virus (HCV). Viral clearance is associated with vigorous and multi-specific CD4(+) T-cell responses, while chronic infection has been shown to be associated with weak or absent T-cell responses. Most of these studies have used functional assays to analyze virus-specific CD4(+) T-cell responses; however, these and other detection methods have various limitations. Therefore, the important question of whether virus-specific CD4(+) T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analyzed in detail. A novel assay, in which virus-specific CD4(+) T-cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to viral antigens, can help to overcome some of the limitations of functional assays and restrictions of multimer-based methods. This and other current established methods for the detection of HCV-specific CD4(+) T cells will be discussed in this review.

Miranda-Novales G, Arriaga-Pizano L, Herrera-Castillo C, Pastelin-Palacios R, Valero-Pacheco N, Pérez-Toledo M, Ferat-Osorio E, Solórzano-Santos F et al. 2015. Antibody responses to influenza viruses in paediatric patients and their contacts at the onset of the 2009 pandemic in Mexico. J Infect Dev Ctries, 9 (3), pp. 259-266. | Show Abstract | Read more

INTRODUCTION: On April 2009, the Mexican Ministry of Health received notification of cases of severe pneumonia mostly affecting young healthy people; this was the beginning of the first influenza pandemic of the 21st century. The nature of the immune response to the influenza A(H1N1)2009 pandemic strain in Mexico at the beginning of the pandemic outbreak has not been completely defined. We describe the serological response to the 2009 pandemic influenza virus in paediatric patients with influenza-like illness, their household contacts (HHCs), and exposed health-care workers (HCWs) at the beginning of the pandemic outbreak in Mexico City. METHODOLOGY: thirty pre-epidemic and 129 epidemic samples were collected and serum antibodies were measured against A(H1N1)2009 pandemic virus and two non-pandemic swine influenza viruses by an haemagglutination inhibition assay . RESULTS: 91% (29/32) of the convalescence samples from confirmed patients had an antibody titre ≥ 10 (GMT 25), 63% (41/65) of the HHCs (GMT 12), 41% of HCWs (GMT 6) and 13% (4/30) of pre-epidemic samples (GMT 6) for the pandemic influenza virus. Of the 32 confirmed cases, 60% had an antibody titre ≥ 40 for the pandemic strain, 53% for the A/swine/Iowa(H1N1) virus (GMT 62) and 43% for the A/swine/Texas(H3N2) virus (GMT 66). CONCLUSION: The antibody response to 2009 pandemic influenza virus was widespread in convalescence samples from patients with confirmed pandemic influenza infection but the GMT was below the protective titre. There was no evidence that antibodies to the swine influenza viruses had cross-protective effect against the 2009 pandemic influenza virus.

Matthews PC, Jeffery K, Klenerman P, Barnes E, Cooke G. 2015. Screening and treatment for hepatitis C: a balanced perspective. BMJ, 350 (feb24 10), pp. h644. | Read more

Webster DP, Klenerman P, Dusheiko GM. 2015. Hepatitis C The Lancet, | Show Abstract | Read more

© 2015 Elsevier Ltd. Hepatitis C virus (HCV) infection is a major health problem worldwide. The effects of chronic infection include cirrhosis, end-stage liver disease, and hepatocellular carcinoma. As a result of shared routes of transmission, co-infection with HIV is a substantial problem, and individuals infected with both viruses have poorer outcomes than do peers infected with one virus. No effective vaccine exists, although persistent HCV infection is potentially curable. The standard of care has been subcutaneous interferon alfa and oral ribavirin for 24-72 weeks. This treatment results in a sustained virological response in around 50% of individuals, and is complicated by clinically significant adverse events. In the past 10 years, advances in HCV cell culture have enabled an improved understanding of HCV virology, which has led to development of many new direct-acting antiviral drugs that target key components of virus replication. These direct-acting drugs allow for simplified and shortened treatments for HCV that can be given as oral regimens with increased tolerability and efficacy than interferon and ribavirin. Remaining obstacles include access to appropriate care and treatment, and development of a vaccine.

Sims S, Klenerman P. 2015. Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells European Journal of Immunology, 45 (1), pp. 113-118. | Show Abstract | Read more

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Murine CMV (MCMV) infection induces effector CD8+ T cells that continue to increase in frequency after acute infection ("inflation") and are stably maintained at a high frequency, with up to 20% of the CD8+ T-cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8+ T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8+ T cells. Treatment of MCMV-infected mice with MHC-Class I-saporin tetramers led to partial (80-90%) depletion of epitope-specific CD8+ T cells-rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8+ T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the "inflated" CD8+ T cell responses during persistent infection.

Klenerman P, Ramamurthy N. 2015. Liver sinusoidal endothelial cells: an antiviral "defendothelium". Gastroenterology, 148 (2), pp. 288-291. | Read more

Fergusson JR, Smith KE, Fleming VM, Rajoriya N, Newell EW, Simmons R, Marchi E, Björkander S et al. 2014. CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages. Cell Rep, 9 (3), pp. 1075-1088. | Show Abstract | Read more

The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage.

Swadling L, Capone S, Antrobus RD, Brown A, Richardson R, Newell EW, Halliday J, Kelly C et al. 2014. A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory. Sci Transl Med, 6 (261), pp. 261ra153. | Show Abstract | Read more

A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8(+) and CD4(+) HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.

Sims S, Bolinger B, Klenerman P. 2015. Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells. Eur J Immunol, 45 (1), pp. 113-118. | Show Abstract | Read more

Murine CMV (MCMV) infection induces effector CD8(+) T cells that continue to increase in frequency after acute infection ("inflation") and are stably maintained at a high frequency, with up to 20% of the CD8(+) T-cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8(+) T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8(+) T cells. Treatment of MCMV-infected mice with MHC-Class I-saporin tetramers led to partial (80-90%) depletion of epitope-specific CD8(+) T cells-rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8(+) T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the "inflated" CD8(+) T cell responses during persistent infection.

Xue L, Fergusson J, Salimi M, Panse I, Ussher JE, Hegazy AN, Vinall SL, Jackson DG et al. 2015. Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk. J Allergy Clin Immunol, 135 (5), pp. 1358-66.e1-11. | Show Abstract | Read more

BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K, Kang YH, Walker LJ, Hansen TH, Willberg CB, Klenerman P. 2015. MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets. Mucosal Immunol, 8 (2), pp. 429-440. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1. MAIT cells are activated by a broad range of bacteria through detection of riboflavin metabolites bound by MR1, but their direct cytolytic capacity upon recognition of cognate target cells remains unclear. We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK. Bacterial activation of MAIT cells rapidly induced GrB and perforin, licensing these cells to kill their cognate target cells. Using a novel flow cytometry-based killing assay, we show that licensed MAIT cells, but not ex vivo MAIT cells from the same donors, can efficiently kill Escherichia coli-exposed B-cell lines in an MR1- and degranulation-dependent manner. Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation. The tightly regulated cytolytic capacity of MAIT cells may have an important role in the control of intracellular bacterial infections, such as Mycobacterium tuberculosis.

Kelly C, Swadling L, Brown A, Capone S, Folgori A, Salio M, Klenerman P, Barnes E. 2015. Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes. Eur J Immunol, 45 (1), pp. 309-316. | Show Abstract | Read more

Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS3(1073) and NS3(1446), and multiple variants for epitope NS3(1406) that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross-reactivity of vaccine-induced T cells was determined using variant peptides in IFN-γ ELISPOT assays. Vaccine-induced T cells targeted approximately 90% of NS3(1073) genotype 1 sequences and 50% of NS3(1446) genotype 1 and 3 sequences. For NS3(1406), 62% of subtype-1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS3(1406) that was maximally cross-reactive. In vitro priming assays showed that of those tested the NS3(1406) vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross-reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross-reactive T cells at immunodominant epitopes. The degree of cross-reactivity varies between epitopes and may be HCV-subtype specific.

Walker LJ, Tharmalingam H, Klenerman P. 2014. The rise and fall of MAIT cells with age. Scand J Immunol, 80 (6), pp. 462-463. | Read more

Iles JC, Raghwani J, Harrison GL, Pepin J, Djoko CF, Tamoufe U, LeBreton M, Schneider BS et al. 2014. Phylogeography and epidemic history of hepatitis C virus genotype 4 in Africa. Virology, 464-465 (1), pp. 233-243. | Show Abstract | Read more

HCV genotype 4 is prevalent in many African countries, yet little is known about the genotype׳s epidemic history on the continent. We present a comprehensive study of the molecular epidemiology of genotype 4. To address the deficit of data from the Democratic Republic of the Congo (DRC) we PCR amplified 60 new HCV isolates from the DRC, resulting in 33 core- and 48 NS5B-region sequences. Our data, together with genotype 4 database sequences, were analysed using Bayesian phylogenetic approaches. We find three well-supported intra-genotypic lineages and estimate that the genotype 4 common ancestor existed around 1733 (1650-1805). We show that genotype 4 originated in central Africa and that multiple lineages have been exported to north Africa since ~1850, including subtype 4a which dominates the epidemic in Egypt. We speculate on the causes of the historical intra-continental spread of genotype 4, including population movements during World War 2.

Lopez-Granados E, Stacey M, Kienzler AK, Sierro S, Willberg CB, Fox CP, Rigaud S, Long HM et al. 2014. A mutation in X-linked inhibitor of apoptosis (G466X) leads to memory inflation of Epstein-Barr virus-specific T cells. Clin Exp Immunol, 178 (3), pp. 470-482. | Show Abstract | Read more

Mutations in the X-linked inhibitor of apoptosis (XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus (EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP (G466X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4(+) CD8(+) population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon (IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAP(G466X) mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease.

Beverley PC, Ruzsics Z, Hey A, Hutchings C, Boos S, Bolinger B, Marchi E, O'Hara G, Klenerman P, Koszinowski UH, Tchilian EZ. 2014. A novel murine cytomegalovirus vaccine vector protects against Mycobacterium tuberculosis. J Immunol, 193 (5), pp. 2306-2316. | Show Abstract | Read more

Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.

Fitzmaurice K, Hurst J, Dring M, Rauch A, McLaren PJ, Günthard HF, Gardiner C, Klenerman P et al. 2014. Additive effects of HLA alleles and innate immune genes determine viral outcome in HCV infection Gut, | Show Abstract | Read more

Background Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. Objective This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. Design We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. Results Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 'T' allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome. Conclusions This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response. © 2014 BMJ Publishing Group Ltd & British Society of Gastroenterology.

Fitzmaurice K, Hurst J, Dring M, Rauch A, McLaren PJ, Günthard HF, Gardiner C, Klenerman P, Irish HCV Research Consortium, Swiss HIV Cohort Study. 2015. Additive effects of HLA alleles and innate immune genes determine viral outcome in HCV infection. Gut, 64 (5), pp. 813-819. | Show Abstract | Read more

BACKGROUND: Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. OBJECTIVE: This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. DESIGN: We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. RESULTS: Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 'T' allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome. CONCLUSIONS: This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response.

Matthews PC, Geretti AM, Goulder PJ, Klenerman P. 2014. Epidemiology and impact of HIV coinfection with hepatitis B and hepatitis C viruses in Sub-Saharan Africa. J Clin Virol, 61 (1), pp. 20-33. | Show Abstract | Read more

Human immunodeficiency virus (HIV), Hepatitis B (HBV) and Hepatitis C (HCV) are blood-borne viruses with potentially shared routes of transmission. In high-income settings, the impact of antiretroviral therapy (ART) on survival has unmasked chronic liver disease from viral hepatitis B or hepatitis C as a leading cause of morbidity and mortality in individuals with HIV infection. It is now feared that progressive liver disease may threaten the success of ART programmes in developing countries, where HCV or HBV testing and monitoring are not yet systematic among HIV-infected patients and ART use is generally blind to these co-infections. We set out to review recent data from Sub-Saharan Africa, in order to build a detailed and up-to-date picture of the epidemiology and emerging impact of HBV and HCV coinfection in countries at the heart of the HIV pandemic. There is a preponderance of HIV/HBV coinfection compared to HIV/HCV in this region, and significant caveats exist regarding the accuracy of published HCV seroprevalence surveys. Morbidity and mortality of coinfection is significant, and may be further enhanced in African populations due to the influence of host, viral and environmental factors. Careful scrutiny of the coinfection problem is vital to inform an approach to directing resources, planning public health initiatives, providing clinical care, and guiding future research.

Kløverpris HN, McGregor R, McLaren JE, Ladell K, Stryhn A, Koofhethile C, Brener J, Chen F et al. 2014. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load. AIDS, 28 (14), pp. 2007-2021. | Show Abstract | Read more

OBJECTIVES: Although CD8+ T cells play a critical role in the control of HIV-1 infection,their antiviral efficacy can be limited by antigenic variation and immune exhaustion.The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. DESIGN AND METHODS: Here, we used an array of different human leukocyte antigen(HLA)-B*15:03 and HLA-B*42:01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations(n = 128) spanning 11 different epitope targets. RESULTS: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. CONCLUSION: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure.

Jo J, Tan AT, Ussher JE, Sandalova E, Tang XZ, Tan-Garcia A, To N, Hong M et al. 2014. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver. PLoS Pathog, 10 (6), pp. e1004210. | Show Abstract | Read more

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright) mucosal-associated invariant T (MAIT) and CD56(Bright) NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

Halliday JS, Harrison GL, Brown A, Hunter JG, Bendall R, Penny D, Toatu T, Abdad MY, Klenerman P, Barnes E, Dalton HR. 2014. Hepatitis E virus infection, Papua New Guinea, Fiji, and Kiribati, 2003-2005. Emerg Infect Dis, 20 (6), pp. 1057-1058. | Read more

Matthews PC, Malik A, Simmons R, Sharp C, Simmonds P, Klenerman P. 2014. PARV4: an emerging tetraparvovirus. PLoS Pathog, 10 (5), pp. e1004036. | Read more

Stacey MA, Marsden M, Pham N TA, Clare S, Dolton G, Stack G, Jones E, Klenerman P et al. 2014. Neutrophils recruited by IL-22 in peripheral tissues function as TRAIL-dependent antiviral effectors against MCMV. Cell Host Microbe, 15 (4), pp. 471-483. | Show Abstract | Read more

During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs.

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55

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Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie ANJ, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells Journal of Allergy and Clinical Immunology, 133 (4), | Show Abstract | Read more

Background Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on T H2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. Objectives We sought to determine the role of PGD2 and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. Methods The effects of PGD 2, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD2 under physiologic conditions were evaluated by using the supernatant from activated mast cells. Results PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD 2 on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Conclusions PGD2 is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s. © 2013 American Academy of Allergy, Asthma & Immunology.

Bapat M. 2014. Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients using proteomics Journal of Molecular Biomarkers & Diagnosis, s5 (01), | Read more

Sims S, Colston J, Bolinger B, Emery V, Klenerman P. 2014. CD73 is dispensable for the regulation of inflationary CD8+ T-cells after murine cytomegalovirus infection and adenovirus immunisation. PLoS One, 9 (12), pp. e114323. | Show Abstract | Read more

The ecto-5'-nucleotidase (CD73) is expressed by T-cell subsets, myeloid derived suppressive cells and endothelial cells. It works in conjunction with CD39 to regulate the formation and degradation of adenosine in vivo. Adenosine has previously been shown to suppress the proliferation and cytokine secretion of T-cells and recent evidence suggests that inhibition of CD73 has the potential to enhance T-cell directed therapies. Here we utilised a CD73 knockout mouse model to assess the suppressive ability of CD73 on CD8+ T-cell classical memory and memory "inflation", induced by murine cytomegalovirus (MCMV) infection and adenovirus immunisation. We show that CD73 is dispensable for normal CD8+ T-cell differentiation and function in both models. Thus CD73 as a suppressor of CD8+ T-cells is unlikely to play a deterministic role in the generation and functional characteristics of antiviral memory in these settings.

Puleston DJ, Zhang H, Powell TJ, Lipina E, Sims S, Panse I, Watson AS, Cerundolo V, Townsend AR, Klenerman P, Simon AK. 2014. Autophagy is a critical regulator of memory CD8(+) T cell formation. Elife, 3 (November), pp. 1-21. | Show Abstract | Read more

During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

Ussher JE, Klenerman P, Willberg CB. 2014. Mucosal-associated invariant T-cells: new players in anti-bacterial immunity. Front Immunol, 5 (OCT), pp. 450. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population involved in anti-bacterial immunity. In human beings, MAIT cells are abundant, comprising ~10% of the CD8(+) T-cell compartment in blood. They are enriched at mucosal sites and are particularly prevalent within the liver. MAIT cells are defined by the expression of a semi-invariant T-cell receptor (Vα7.2-Jα33/12/20) and are restricted by the non-polymorphic, highly evolutionarily conserved MHC class Ib molecule, MHC-related protein (MR)1. MR1 has recently been shown to present an unstable pyrimidine intermediate derived from a biosynthetic precursor of riboflavin; riboflavin biosynthesis occurs in many bacteria but not in human beings. Consistent with this, MAIT cells are responsive to riboflavin-metabolizing bacteria, including Salmonella. In mouse models, MAIT cells have been shown to play a non-redundant role in anti-bacterial immunity, including against Escherichia coli, Klebsiella pneumoniae, and Mycobacterium bovis BCG. In human beings, MAIT cells are decreased in frequency in the blood of patients with tuberculosis or pneumonia, and their frequency has been inversely correlated with the risk of subsequent systemic bacterial infection in patients in intensive care. Intriguingly, MAIT cells are also depleted from the blood early in HIV infection and fail to recover with anti-retroviral therapy, which may contribute to the susceptibility of patients infected with HIV to certain bacterial infections, including non-typhoidal Salmonella. In this review, we will discuss what is currently known about MAIT cells, the role that Salmonella has played in elucidating MAIT cell restriction and function, and the role MAIT cells might play in the control of Salmonella infection.

Kløverpris HN, McGregor R, McLaren JE, Ladell K, Stryhn A, Koofhethile C, Brener J, Chen F et al. 2014. Programmed death-1 expression on HIV-1-specific CD8<sup>+</sup> T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load AIDS, 28 (14), pp. 2007-2021. | Show Abstract | Read more

Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B*15 : 03 and HLA-B*42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n=128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. © 2014 Wolters Kluwer Health.

Rajoriya N, Fergusson JR, Leithead JA, Klenerman P. 2014. Gamma Delta T-lymphocytes in Hepatitis C and Chronic Liver Disease. Front Immunol, 5 (AUG), pp. 400. | Show Abstract | Read more

Discovered 30 years ago, gamma delta (γδ) T-lymphocytes remain an intriguing and enigmatic T-cell subset. Although in humans they comprise a small fraction of the total circulating T-lymphocyte pool, they represent an important T-cell subset in tissues such as the liver, with roles bridging the innate and adaptive immune systems. The associations of γδ T-lymphocytes with chronic liver disease have been explored - however, there remain conflicting data as to whether these T-cells are pathogenic or protective. In patients with some forms of liver disease, their expansion in the periphery and especially in the liver may indeed help pathogen clearance, while in other conditions their presence may, in contrast, contribute to disease progression. γδ T-cells can also express CD161, a C-type lectin, and such cells have been found to be involved in the pathogenesis of inflammatory disease. CD161+ T-cells of diverse subsets are known to be enriched in the livers of patients with chronic hepatitis C. This article serves to provide a review of the γδ T-cell population and its role in hepatitis C and other chronic liver diseases, and also explores a potential role of the CD161+ γδ T-cells in liver diseases.

Cited:

25

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Matthews PC, Geretti AM, Goulder PJR, Klenerman P. 2014. Epidemiology and impact of HIV coinfection with Hepatitis B and Hepatitis C viruses in Sub-Saharan Africa Journal of Clinical Virology, 61 (1), pp. 20-33. | Show Abstract | Read more

Human immunodeficiency virus (HIV), Hepatitis B (HBV) and Hepatitis C (HCV) are blood-borne viruses with potentially shared routes of transmission. In high-income settings, the impact of antiretroviral therapy (ART) on survival has unmasked chronic liver disease from viral hepatitis B or hepatitis C as a leading cause of morbidity and mortality in individuals with HIV infection. It is now feared that progressive liver disease may threaten the success of ART programmes in developing countries, where HCV or HBV testing and monitoring are not yet systematic among HIV-infected patients and ART use is generally blind to these co-infections. We set out to review recent data from Sub-Saharan Africa, in order to build a detailed and up-to-date picture of the epidemiology and emerging impact of HBV and HCV coinfection in countries at the heart of the HIV pandemic. There is a preponderance of HIV/HBV coinfection compared to HIV/HCV in this region, and significant caveats exist regarding the accuracy of published HCV seroprevalence surveys. Morbidity and mortality of coinfection is significant, and may be further enhanced in African populations due to the influence of host, viral and environmental factors. Careful scrutiny of the coinfection problem is vital to inform an approach to directing resources, planning public health initiatives, providing clinical care, and guiding future research. © 2014 Elsevier B.V.

Williams SK, Donaldson E, Van Der Kleij T, Dixon L, Fisher M, Tibble J, Gilleece Y, Klenerman P, Banham AH, Howard M, Webster DP. 2014. Quantification of hepatic FOXP3+ T-lymphocytes in HIV/hepatitis C coinfection Journal of Viral Hepatitis, 21 (4), pp. 251-259. | Show Abstract | Read more

Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T-regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P = 0.031) and CD4+ cells (P = 0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P = 0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P < 0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients. © 2013 John Wiley & Sons Ltd.

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Scopus

Wong EB, Akilimali NA, Govender P, Sullivan ZA, Cosgrove C, Pillay M, Lewinsohn DM, Bishai WR et al. 2013. Low levels of peripheral CD161++CD8+ Mucosal Associated Invariant T (MAIT) cells are found in HIV and HIV/TB co-infection PLoS ONE, 8 (12), | Show Abstract | Read more

Background: High expression of CD161 on CD8+ T cells is associated with a population of cells thought to play a role in mucosal immunity. We wished to investigate this subset in an HIV and Mycobacterium tuberculosis (MTB) endemic African setting. Methods: A flow cytometric approach was used to assess the frequency and phenotype of CD161++CD8+ T cells. 80 individuals were recruited for cross-sectional analysis: controls (n = 18), latent MTB infection (LTBI) only (n = 16), pulmonary tuberculosis (TB) only (n = 8), HIV only (n = 13), HIV and LTBI co-infection (n = 15) and HIV and TB co-infection (n = 10). The impact of acute HIV infection was assessed in 5 individuals recruited within 3 weeks of infection. The frequency of CD161++CD8+ T cells was assessed prior to and during antiretroviral therapy (ART) in 14 HIV-positive patients. Results: CD161++CD8+ T cells expressed high levels of the HIV co-receptor CCR5, the tissue-homing marker CCR6, and the Mucosal-Associated Invariant T (MAIT) cell TCR Vα7.2. Acute and chronic HIV were associated with lower frequencies of CD161++CD8+ T cells, which did not correlate with CD4 count or HIV viral load. ART was not associated with an increase in CD161++CD8+ T cell frequency. There was a trend towards lower levels of CD161++CD8+ T cells in HIV-negative individuals with active and latent TB. In those co-infected with HIV and TB, CD161++CD8+ T cells were found at low levels similar to those seen in HIV mono-infection. Conclusions: The frequencies and phenotype of CD161++CD8+ T cells in this South African cohort are comparable to those published in European and US cohorts. Low-levels of this population were associated with acute and chronic HIV infection. Lower levels of the tissue-trophic CD161++CD8+ T cell population may contribute to weakened mucosal immune defense, making HIV-infected subjects more susceptible to pulmonary and gastrointestinal infections and detrimentally impacting on host defense against TB.

Xue L, Salimi M, Panse I, Mjösberg JM, McKenzie AN, Spits H, Klenerman P, Ogg G. 2014. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells. J Allergy Clin Immunol, 133 (4), pp. 1184-1194. | Show Abstract | Read more

BACKGROUND: Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2), a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. OBJECTIVES: We sought to determine the role of PGD₂ and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. METHODS: The effects of PGD₂, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD₂ under physiologic conditions were evaluated by using the supernatant from activated mast cells. RESULTS: PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD₂ on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. CONCLUSIONS: PGD₂ is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.

Newey PJ, Gorvin CM, Cleland SJ, Willberg CB, Bridge M, Azharuddin M, Drummond RS, van der Merwe PA, Klenerman P, Bountra C, Thakker RV. 2013. Mutant prolactin receptor and familial hyperprolactinemia. N Engl J Med, 369 (21), pp. 2012-2020. | Show Abstract | Read more

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

Iles JC, Abby Harrison GL, Lyons S, Djoko CF, Tamoufe U, Lebreton M, Schneider BS, Fair JN et al. 2013. Hepatitis C virus infections in the Democratic Republic of Congo exhibit a cohort effect. Infect Genet Evol, 19 pp. 386-394. | Show Abstract | Read more

The prevalence and genetic diversity of hepatitis C virus (HCV) and human pegivirus (HPgV) in many regions of sub-Saharan Africa is poorly characterized, including in the Democratic Republic of Congo--the largest country in the region and one of the most populous. To address this situation we conducted a molecular epidemiological survey of HCV and HPgV (previously named GB Virus C or hepatitis G virus) in samples collected in 2007 from 299 males from the DRC, whose ages ranged from 21 to 71 years old. Samples were tested for the presence of HCV antibodies by ELISA and reactive samples were subsequently tested for HCV RNA using RT-PCR in which both the HCV Core and NS5B genome regions were amplified. Remaining samples were tested for HPgV RNA and the HPgV NS3 genome region of positive samples was amplified. For HCV, 13.7% of the samples were seropositive (41/299) but only 3.7% were viremic (11/299). HPgV RNA was found in 12.7% (33/259) of samples. HCV viremia was strongly associated with age; the percentage of samples that contained detectable HCV RNA was ~0.5% in those younger than 50 and 13% in those older than 50. Our study represents the first systematic survey of HCV genetic diversity in the DRC. HCV sequences obtained belonged to diverse lineages of genotype 4, including subtypes 4c, 4 k, 4 l and 4r, plus one unclassified lineage that may constitute a new subtype. These data suggest that HCV in the DRC exhibits an age 'cohort effect', as has been recently reported in neighbouring countries, and are consistent with the hypothesis that HCV transmission rates were higher in the mid-twentieth century, possibly as a result of parenteral, iatrogenic, or other unidentified factors. Different HCV subtypes were associated with individuals of different ages, implying that HCV infection in the DRC may have arisen through multiple separate HCV epidemics with different causes.

Colston J, Bolinger B, Turner A, Gilbert S, Klenerman P. 2013. AN ADENOVIRAL MODEL TO UNLOCK THE SECRETS OF MEMORY INFLATION? JOURNAL OF INFECTION, 67 (4), pp. 345-345. | Read more

Fitzmaurice K, Hurst J, Dring M, Gardiner C, Klenerman P, Consortium IHR. 2013. HLA ALLELES IN COMBINATION WITH INNATE IMMUNE GENES ARE KEY DETERMINANTS OF VIRAL OUTCOME IN HEPATITIS C VIRUS INFECTION JOURNAL OF INFECTION, 67 (4), pp. 345-345. | Read more

Ussher JE, Bilton M, Attwod E, Shadwell J, Richardson R, de Lara C, Mettke E, Kurioka A, Hansen TH, Klenerman P, Willberg CB. 2014. CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner. Eur J Immunol, 44 (1), pp. 195-203. | Show Abstract | Read more

CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-α chain 7.2 (Vα7.2)-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) Vα7.2(+) and CD161(++) Vα7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.

Batty EM, Wong TH, Trebes A, Argoud K, Attar M, Buck D, Ip CL, Golubchik T et al. 2013. A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples. PLoS One, 8 (6), pp. e66129. | Show Abstract | Read more

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.

Rowland R, O'Hara GA, Hamill M, Poulton ID, Donaldson H, Dinsmore L, James T, Barnes E et al. 2013. Determining the validity of hospital laboratory reference intervals for healthy young adults participating in early clinical trials of candidate vaccines. Hum Vaccin Immunother, 9 (8), pp. 1741-1751. | Show Abstract | Read more

This was a retrospective study to determine the validity of institutional reference intervals for interpreting biochemistry and hematology results in healthy adults in the context of clinical trials of preventive vaccines. An example population of 974 healthy adults participating in clinical trials at the Jenner Institute, Oxford, UK, between 1999 and 2009 was studied. Methods for calculating the central 95% ranges and determining the coefficients of within person variation were demonstrated. Recommendations have been made as to how these data can be usefully applied to the interpretation of blood results in healthy adult subjects for the purposes of clinical trial inclusion decisions and post-vaccination safety monitoring.

Griffiths SJ, Riddell NE, Masters J, Libri V, Henson SM, Wertheimer A, Wallace D, Sims S et al. 2013. Age-associated increase of low-avidity cytomegalovirus-specific CD8+ T cells that re-express CD45RA. J Immunol, 190 (11), pp. 5363-5372. | Show Abstract | Read more

The mechanisms regulating memory CD8(+) T cell function and homeostasis during aging are unclear. CD8(+) effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8(+) T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8(+) T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8(+) T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA(-)CD45RO(+) CMV-specific CD8(+) T cells induced CD45RA expression while Ag activated cells remained CD45RO(+). This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8(+)CD45RA(+) T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.

Taylor MJ, Godlewska B, Near J, Christmas D, Potokar J, Collier J, Klenerman P, Barnes E, Cowen PJ. 2014. Effect of interferon-α on cortical glutamate in patients with hepatitis C: a proton magnetic resonance spectroscopy study. Psychol Med, 44 (4), pp. 789-795. | Show Abstract | Read more

BACKGROUND: The development of depressive symptomatology is a recognized complication of treatment with the cytokine interferon-α (IFN-α) and has been seen as supporting inflammatory theories of the pathophysiology of major depression. Major depression has been associated with changes in glutamatergic activity and recent formulations of IFN-induced depression have implicated neurotoxic influences that could also lead to changes in glutamate function. The present study used magnetic resonance spectroscopy (MRS) to measure glutamate and its major metabolite glutamine in patients with hepatitis C who received treatment with pegylated IFN-α and ribavirin. METHOD: MRS measurements of glutamate and glutamine were taken from a 25 × 20 × 20 mm voxel including the pregenual anterior cingulate cortex in 12 patients before and after 4-6 weeks of treatment with IFN. RESULTS: IFN treatment led to an increase in cortical levels of glutamine (p = 0.02) and a significant elevation in the ratio of glutamine to glutamate (p < 0.01). Furthermore, changes in glutamine level correlated significantly with ratings of depression and anxiety at the time of the second scan. CONCLUSIONS: We conclude that treatment with IFN-α is associated with MRS-visible changes in glutamatergic metabolism. However, the changes seen differ from those reported in major depression, which suggests that the pathophysiology of IFN-induced depression may be distinct from that of major depression more generally.

Swadling L, Klenerman P, Barnes E. 2013. Ever closer to a prophylactic vaccine for HCV. Expert Opin Biol Ther, 13 (8), pp. 1109-1124. | Show Abstract | Read more

INTRODUCTION: With 3 - 4 million new infections occurring annually, hepatitis C virus (HCV) is a major global health problem. There is increasing evidence to suggest that HCV will be highly amenable to a vaccine approach, and despite advances in treatment, a vaccine remains the most cost-effective and realistic means to significantly reduce the worldwide mortality and morbidity associated with persistent HCV infection. AREAS COVERED: In this review we discuss immune responses to HCV during natural infection, and describe how they may inform vaccine design. We introduce the current candidate vaccines for HCV and compare how these fare against the expected requirements of an effective prophylactic HCV vaccine in relation to the breadth, functionality, magnitude and phenotype of the vaccine-induced immune response. EXPERT OPINION: Although the correlates of immune protection against HCV are not completely defined, we now have vaccine technologies capable of inducing HCV-specific adaptive immune responses to an order of magnitude that are associated with protection during natural infection. The challenge next is to i) establish well-characterised cohorts of people at risk of HCV infection for vaccine efficacy testing and ii) to better understand the correlates of protection in natural history studies. If these can be achieved, a vaccine against HCV appears a realistic goal.

Williams S, Donaldson E, Van der Kleij T, Dixon L, Fisher M, Tibble J, Gilleece Y, Klenerman P, Banham A, Howard M, Webster D. 2013. Quantification of hepatic FOXP3+ T-lymphocytes in HIV/hepatitis C co-infection - a mechanism for poor outcomes? HIV MEDICINE, 14 pp. 4-4.

Bolinger B, Sims S, O'Hara G, de Lara C, Tchilian E, Firner S, Engeler D, Ludewig B, Klenerman P. 2013. A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector. J Immunol, 190 (8), pp. 4162-4174. | Show Abstract | Read more

CD8(+) T cell memory inflation, first described in murine CMV (MCMV) infection, is characterized by the accumulation of high-frequency, functional Ag-specific CD8(+) T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of Ag is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus's low-level persistence and stochastic reactivation. We developed a new model of memory inflation based on a β-galactosidase (βgal)-recombinant adenovirus vector. After i.v. administration in C57BL/6 mice, we observed marked memory inflation in the βgal96 epitope, whereas a second epitope, βgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype, and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC class II. As in MCMV, only the inflating epitope showed immunoproteasome independence. These data define a new model for memory inflation, which is fully replication independent, internally controlled, and reproduces the key immunologic features of the CD8(+) T cell response. This model provides insight into the mechanisms responsible for memory inflation and, because it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans.

Bucci C, von Delft A, Christian A, Flemming VM, Harrison A, Halliday J, Collier J, Manganis C, Klenerman P, Irving W, Barnes E. 2013. 'Favourable' IL28B polymorphisms are associated with a marked increase in baseline viral load in hepatitis C virus subtype 3a infection and do not predict a sustained virological response after 24 weeks of therapy. J Gen Virol, 94 (Pt 6), pp. 1259-1265. | Show Abstract | Read more

IL28B host genetic make-up is known to play a critical role in the outcome of genotype 1 hepatitis C virus (HCV) infection in the context of both primary infection and therapy. However, the role of IL28B in subtype 3a infection remains unclear, and has not yet been assessed in the UK population where subtype 3a is dominant. In this study, we evaluated the role of the IL28B single-nucleotide polymorphism rs8099917 in 201 patients recruited from two well-defined cohorts (from Nottingham and Oxford), treated with the standard-of-care therapy of pegylated interferon and ribavirin for 24 weeks. We showed that the 'favourable' IL28B gene was associated with a rapid virological response to therapy at 4 weeks (P<0.0001), but not with a sustained virological response to therapy. The median viral load at baseline, before therapy, was markedly increased in people with the 'favourable' IL28B genotype [median viral load for the TT allele, 925,961 IU ml(-1) (range 2200-21,116,965 IU ml(-1)), and for the GT or GG allele, 260,284 IU ml(-1) (range 740-7,560,000 IU ml(-1)); P = 0.0010]. Our results suggest that the host genetic response plays an important role in early viral clearance of subtype 3a virus from the blood. However, significant reservoirs of infection must persist, as viral relapse is common, even in those with the favourable host genotype.

Cosgrove C, Ussher JE, Rauch A, Gaertner K, Kurioka A, Huehn MH, Adelmann K, Kang Y-H et al. 2013. EARLY AND NON-REVERSIBLE DECREASE OF CD161++/MUCOSAL ASSOCIATED INVARIANT T CELLS IN HIV INFECTION JOURNAL OF INFECTION, 67 (4), pp. 342-343. | Read more

Cosgrove C, Ussher JE, Rauch A, Gärtner K, Kurioka A, Hühn MH, Adelmann K, Kang YH et al. 2013. Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection. Blood, 121 (6), pp. 951-961. | Show Abstract | Read more

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.

Simmons R, Sharp C, Levine J, Bowness P, Simmonds P, Cox A, Klenerman P. 2013. Evolution of CD8+ T cell responses after acute PARV4 infection. J Virol, 87 (6), pp. 3087-3096. | Show Abstract | Read more

PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2-peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.

Wong EB, Akilimali NA, Govender P, Sullivan ZA, Cosgrove C, Pillay M, Lewinsohn DM, Bishai WR et al. 2013. Low levels of peripheral CD161++CD8+ mucosal associated invariant T (MAIT) cells are found in HIV and HIV/TB co-infection. PLoS One, 8 (12), pp. e83474. | Show Abstract | Read more

BACKGROUND: High expression of CD161 on CD8+ T cells is associated with a population of cells thought to play a role in mucosal immunity. We wished to investigate this subset in an HIV and Mycobacterium tuberculosis (MTB) endemic African setting. METHODS: A flow cytometric approach was used to assess the frequency and phenotype of CD161++CD8+ T cells. 80 individuals were recruited for cross-sectional analysis: controls (n = 18), latent MTB infection (LTBI) only (n = 16), pulmonary tuberculosis (TB) only (n = 8), HIV only (n = 13), HIV and LTBI co-infection (n = 15) and HIV and TB co-infection (n = 10). The impact of acute HIV infection was assessed in 5 individuals recruited within 3 weeks of infection. The frequency of CD161++CD8+ T cells was assessed prior to and during antiretroviral therapy (ART) in 14 HIV-positive patients. RESULTS: CD161++CD8+ T cells expressed high levels of the HIV co-receptor CCR5, the tissue-homing marker CCR6, and the Mucosal-Associated Invariant T (MAIT) cell TCR Vα7.2. Acute and chronic HIV were associated with lower frequencies of CD161++CD8+ T cells, which did not correlate with CD4 count or HIV viral load. ART was not associated with an increase in CD161++CD8+ T cell frequency. There was a trend towards lower levels of CD161++CD8+ T cells in HIV-negative individuals with active and latent TB. In those co-infected with HIV and TB, CD161++CD8+ T cells were found at low levels similar to those seen in HIV mono-infection. CONCLUSIONS: The frequencies and phenotype of CD161++CD8+ T cells in this South African cohort are comparable to those published in European and US cohorts. Low-levels of this population were associated with acute and chronic HIV infection. Lower levels of the tissue-trophic CD161++ CD8+ T cell population may contribute to weakened mucosal immune defense, making HIV-infected subjects more susceptible to pulmonary and gastrointestinal infections and detrimentally impacting on host defense against TB.

Harrison GL, Pryor J, Malani J, Supuri M, Masta A, Teriboriki B, Toatu T, Penny D et al. 2013. Infection frequency of hepatitis C virus and IL28B haplotypes in Papua New Guinea, Fiji, and Kiribati. PLoS One, 8 (8), pp. e66749. | Show Abstract | Read more

It has been estimated that there are more than 60 million Hepatitis C virus (HCV) carriers in the World Health Organisation's Western Pacific region (WHO-WPR), where liver cancer is among the top three causes of cancer death. WHO and the US Centres for Disease Control and Prevention report the prevalence of HCV in the South Pacific islands (countries within the WHO-WPR) to be high (5-10% and >2% respectively). However, since HCV is not tested for in many of these countries, there is sparse data available to support this assertion. We screened ∼2000 apparently healthy individuals from Papua New Guinea, Fiji and Kiribati and found a sero-prevalence of 2.0%, 0.1% and 0%, respectively. All sero-positive samples tested negative for HCV RNA. Curious as to why all the sero-positive individuals were negative for HCV-RNA, we also screened them for the HCV protective IL28B SNP markers rs12979860 and rs8099917. All antibody-positive participants bar one had HCV protective haplotypes. Our results suggest that HCV is present in these Pacific island countries, albeit at a prevalence lower than previous estimates. As none of our participants had undergone antiviral treatment, and therefore must have cleared infection naturally, we hypothesise that genotypes 1 and/or 4 are circulating in South Pacific Island people and that these peoples are genetically predisposed to be more likely to spontaneous resolve HCV infection than to become chronic carriers.

Williams SK, Donaldson E, Van der Kleij T, Dixon L, Fisher M, Tibble J, Gilleece Y, Klenerman P, Banham AH, Howard M, Webster DP. 2014. Quantification of hepatic FOXP3+ T-lymphocytes in HIV/hepatitis C coinfection. J Viral Hepat, 21 (4), pp. 251-259. | Show Abstract | Read more

Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T-regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P = 0.031) and CD4+ cells (P = 0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P = 0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P < 0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients.

Walker LJ, Marrinan E, Muenchhoff M, Ferguson J, Kloverpris H, Cheroutre H, Barnes E, Goulder P, Klenerman P. 2013. CD8αα Expression Marks Terminally Differentiated Human CD8+ T Cells Expanded in Chronic Viral Infection. Front Immunol, 4 (AUG), pp. 223. | Show Abstract | Read more

The T cell co-receptor CD8αβ enhances T cell sensitivity to antigen, however studies indicate CD8αα has the converse effect and acts as a co-repressor. Using a combination of Thymic Leukemia (TL) antigen tetramer, which directly binds CD8αα, anti-CD161, and anti-Vα7.2 antibodies we have been able for the first time to clearly define CD8αα expression on human CD8 T cells subsets. In healthy controls CD8αα is most highly expressed by CD161 "bright" (CD161++) mucosal associated invariant T (MAIT) cells, with CD8αα expression highly restricted to the TCR Vα7.2+ cells of this subset. We also identified CD8αα-expressing populations within the CD161 "mid" (CD161+) and "negative" (CD161-) non-MAIT CD8 T cell subsets and show TL-tetramer binding to correlate with expression of CD8β at low levels in the context of maintained CD8α expression (CD8α+CD8β(low)). In addition, we found CD161-CD8α+CD8β(low) populations to be significantly expanded in the peripheral blood of HIV-1 and hepatitis B (mean of 47 and 40% of CD161- T cells respectively) infected individuals. Such CD8αα expressing T cells are an effector-memory population (CD45RA-, CCR7-, CD62L-) that express markers of activation and maturation (HLA-DR+, CD28-, CD27-, CD57+) and are functionally distinct, expressing greater levels of TNF-α and IFN-γ on stimulation and perforin at rest than their CD8α+CD8β(high) counterparts. Antigen-specific T cells in HLA-B(∗)4201+HIV-1 infected patients are found within both the CD161-CD8α+CD8β(high) and CD161-CD8α+CD8β(low) populations. Overall we have clearly defined CD8αα expressing human T cell subsets using the TL-tetramer, and have demonstrated CD161-CD8α+CD8β(low) populations, highly expanded in disease settings, to co-express CD8αβ and CD8αα. Co-expression of CD8αα on CD8αβ T cells may impact on their overall function in vivo and contribute to the distinctive phenotype of highly differentiated populations in HBV and HIV-1 infection.

Wang H, Yuan Z, Barnes E, Yuan M, Li C, Fu Y, Xia X, Li G et al. 2013. Eight novel hepatitis c virus genomes reveal the changing taxonomic structure of genotype 6 Journal of General Virology, 94 (PART11), pp. 76-80. | Show Abstract | Read more

Analysis of partial hepatitis C virus sequences has revealed many novel genotype 6 variants that cannot be unambiguously classified, which obscure the distinctiveness of pre-existing subtypes. To explore this uncertainty, we obtained genomes of 98.0-98.8% full-length for eight such variants (KM35, QC273, TV257, TV476, TV533, L349, QC271 and DH027) and characterized them using phylogenetic analyses and per cent nucleotide similarities. The former four are closely related phylogenetically to subtype 6k, TV533 and L349 to subtype 6l, QC271 to subtypes 6i and 6j, and DH027 to subtypes 6m and 6n. The former six defined a high-level grouping that comprised subtypes 6k and 6l, plus related strains. The threshold between intra- and intersubtype diversity in this group was indistinct. We propose that similar results would be seen elsewhere if more intermediate variants like QC271 and DH027 were sampled. © 2013 SGM.

Schmidt J, Iversen AK, Tenzer S, Gostick E, Price DA, Lohmann V, Distler U, Bowness P et al. 2012. Rapid antigen processing and presentation of a protective and immunodominant HLA-B*27-restricted hepatitis C virus-specific CD8+ T-cell epitope. PLoS Pathog, 8 (11), pp. e1003042. | Show Abstract | Read more

HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.

Wang H, Yuan Z, Barnes E, Yuan M, Li C, Fu Y, Xia X, Li G et al. 2013. Eight novel hepatitis C virus genomes reveal the changing taxonomic structure of genotype 6. J Gen Virol, 94 (Pt 1), pp. 76-80. | Show Abstract | Read more

Analysis of partial hepatitis C virus sequences has revealed many novel genotype 6 variants that cannot be unambiguously classified, which obscure the distinctiveness of pre-existing subtypes. To explore this uncertainty, we obtained genomes of 98.0-98.8% full-length for eight such variants (KM35, QC273, TV257, TV476, TV533, L349, QC271 and DH027) and characterized them using phylogenetic analyses and per cent nucleotide similarities. The former four are closely related phylogenetically to subtype 6k, TV533 and L349 to subtype 6l, QC271 to subtypes 6i and 6j, and DH027 to subtypes 6m and 6n. The former six defined a high-level grouping that comprised subtypes 6k and 6l, plus related strains. The threshold between intra- and inter-subtype diversity in this group was indistinct. We propose that similar results would be seen elsewhere if more intermediate variants like QC271 and DH027 were sampled.

Prendergast AJ, Klenerman P, Goulder PJ. 2012. The impact of differential antiviral immunity in children and adults. Nat Rev Immunol, 12 (9), pp. 636-648. | Show Abstract | Read more

The course of immune maturation has evolved to favour survival at each stage of development in early life. Fetal and neonatal immune adaptations facilitate intrauterine survival and provide early postnatal protection against extracellular pathogens, but they leave infants susceptible to intracellular pathogens such as viruses that are acquired perinatally. This Review focuses on three such pathogens--HIV, hepatitis B virus and cytomegalovirus--and relates the differential impact of these infections in infants and adults to the antiviral immunity that is generated at different ages. A better understanding of age-specific antiviral immunity may inform the development of integrated prevention, treatment and vaccine strategies to minimize the global disease burden resulting from these infections.

Farci P, Wollenberg K, Diaz G, Engle RE, Lai ME, Klenerman P, Purcell RH, Pybus OG, Alter HJ. 2012. Profibrogenic chemokines and viral evolution predict rapid progression of hepatitis C to cirrhosis. Proc Natl Acad Sci U S A, 109 (36), pp. 14562-14567. | Show Abstract | Read more

Chronic hepatitis C may follow a mild and stable disease course or progress rapidly to cirrhosis and liver-related death. The mechanisms underlying the different rates of disease progression are unknown. Using serial, prospectively collected samples from cases of transfusion-associated hepatitis C, we identified outcome-specific features that predict long-term disease severity. Slowly progressing disease correlated with an early alanine aminotransferase peak and antibody seroconversion, transient control of viremia, and significant induction of IFN-γ and MIP-1β, all indicative of an effective, albeit insufficient, adaptive immune response. By contrast, rapidly progressive disease correlated with persistent and significant elevations of alanine aminotransferase and the profibrogenic chemokine MCP-1 (CCL-2), greater viral diversity and divergence, and a higher rate of synonymous substitution. This study suggests that the long-term course of chronic hepatitis C is determined early in infection and that disease severity is predicted by the evolutionary dynamics of hepatitis C virus and the level of MCP-1, a chemokine that appears critical to the induction of progressive fibrogenesis and, ultimately, the ominous complications of cirrhosis.

Oo YH, Banz V, Kavanagh D, Liaskou E, Withers DR, Humphreys E, Reynolds GM, Lee-Turner L et al. 2012. CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver. J Hepatol, 57 (5), pp. 1044-1051. | Show Abstract | Read more

BACKGROUND & AIMS: IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. METHODS: The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. RESULTS: IL-17(+) T cells comprised 1-3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17(+) cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. CONCLUSIONS: CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

Liaskou E, Withers D, Humphreys E, Shaw JC, Tuohey L, Klenerman P, Adams DH, Oo YH. 2012. KUPFFER CELL DERIVED INTERLEUKIN (IL)-18 INDUCES HEPATIC INFLAMMATION BY PROMOTING LYMPHOCYTE SUBSETS RECRUITMENT ON HEPATIC ENDOTHELIAL CELLS GUT, 61 (Suppl 2), pp. A121-A122. | Read more

Campbell J, Trgovcich J, Kincaid M, Zimmerman PD, Klenerman P, Sims S, Cook CH. 2012. Transient CD8-memory contraction: a potential contributor to latent cytomegalovirus reactivation. J Leukoc Biol, 92 (5), pp. 933-937. | Show Abstract | Read more

It is clear that latent CMV can reactivate in immunocompetent individuals, but the mechanism triggering such reactivations remains unclear. Recent clinical data suggest that reactivation can be subverted by CMV-specific T-memory. We therefore monitored CMV-specific T cells in immunocompetent mice with latent mCMV after a known reactivation trigger (LPS). LPS induced transient systemic contraction of mCMV-specific CD8 memory that was followed by transcriptional reactivation. Subsequent recovery of mCMV-specific T cells coincided with resumption of latency. These data suggest that bacterial antigen encounters can induce transient T-memory contraction, allowing viral recrudescence in hosts latently infected with herpes family viruses.

Gray RR, Salemi M, Klenerman P, Pybus OG. 2012. A new evolutionary model for hepatitis C virus chronic infection. PLoS Pathog, 8 (5), pp. e1002656. | Read more

Simmons R, Sharp C, McClure CP, Rohrbach J, Kovari H, Frangou E, Simmonds P, Irving W et al. 2012. Parvovirus 4 infection and clinical outcome in high-risk populations. J Infect Dis, 205 (12), pp. 1816-1820. | Show Abstract | Read more

Parvovirus 4 (PARV4) is a DNA virus frequently associated with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, but its clinical significance is unknown. We studied the prevalence of PARV4 antibodies in 2 cohorts of HIV- and HCV-infected individuals (n = 469) and the correlations with disease status. We found that PARV4 infection frequently occurred in individuals exposed to bloodborne viruses (95% in HCV-HIV coinfected intravenous drug users [IDUs]). There were no correlations between PARV4 serostatus and HCV outcomes. There was, however, a significant association with early HIV-related symptoms, although because this was tightly linked to both HCV status and clinical group (IDU), the specific role of PARV4 is not yet clear.

Kelly C, Folgori A, Capone S, Brown AC, Swadling L, Townsend R, Halliday J, Stafford E et al. 2012. IN VIVO ANTIGENIC TARGETS OF T CELLS INDUCED BY ADENOVIRAL VECTORED VACCINES IN PATIENTS WITH CHRONIC HCV INFECTION JOURNAL OF HEPATOLOGY, 56 pp. S468-S468.

Kasprowicz VO, Halliday JS, Mitchell J, Klenerman P. 2012. MIGRAs: are they the new IGRAs? Development of monokine-amplified IFN-γ release assays. Biomark Med, 6 (2), pp. 177-186. | Show Abstract | Read more

IFN-γ release by antigen-specific T cells can be used to track immune responses to infections and vaccines. In recent years, there have been substantial advances in the techniques available to measure IFN-γ release and a generation of such assays are now available for clinical use, as well as in a research setting. Interferon release leads to subsequent release of interferon-responsive chemokines such as MIG and IP-10, thus amplifying the original signal. A number of investigators have assessed whether measurement of these chemokines might provide a sensitive platform for detection of infection and antigen-specific T-cell responses. In this article, we assess the potential of these new approaches. We have termed the new antigen-specific T-cell assays monokine-amplified IFN-γ release assays (MIGRAs). Overall, it seems likely that improvements in the detection threshold could be made by analysis of antigen-triggered chemokines and potentially of other molecules in the future, although whether MIGRAs will provide additional clinical utility still remains to be determined.

Phadwal K, Alegre-Abarrategui J, Watson AS, Pike L, Anbalagan S, Hammond EM, Wade-Martins R, McMichael A, Klenerman P, Simon AK. 2012. A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells. Autophagy, 8 (4), pp. 677-689. | Show Abstract | Read more

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.

Humphreys IS, von Delft A, Brown A, Hibbert L, Collier JD, Foster GR, Rahman M, Christian A, Klenerman P, Barnes E. 2012. HCV genotype-3a T cell immunity: specificity, function and impact of therapy. Gut, 61 (11), pp. 1589-1599. | Show Abstract | Read more

BACKGROUND: Hepatitis C virus (HCV) genotype-3a infection is now the dominant strain in South Asia and the UK. Characteristic features include a favourable response to therapy; the reasons for this are unknown but may include distinct genotype-3a-specific T cell immunity. In contrast to genotype-1 infection, T cell immunity to this subtype is poorly defined. OBJECTIVES: The aims of the study were to (1) define the frequency, specificity and cross-reactivity of T cell immunity across the whole viral genome in genotype-3a infection and (2) assess the impact of interferon (IFN)-α/ribavirin on T cell immunity. DESIGN: T cell responses in chronic and resolved HCV genotype-3a were analysed in comparison with genotype-1 infection (total n=85) using specific peptide panels in IFN-γ ELISpot assays. T cell responses were followed longitudinally in a subset of genotype-3a infected patients receiving therapy. Responses were further defined by CD4 and CD8 subset analysis, sequencing of autologous virus and cross-reactivity of genotype-3a with genotype-1a/-1b antigens. RESULTS: CD8 T cell responses commonly targeted the non-structural (NS) proteins in chronic genotype-3a infection whereas in genotype-1 infection CD4 responses targeting HCV core predominated (p=0.0183). Resolved infection was associated with CD4 T cells targeting NS proteins. Paradoxically, a sustained response to therapy was associated with a brisk decline in virus-specific and total lymphocyte counts that recovered after treatment. CONCLUSION: HCV genotype-3a exhibits a distinct T cell specificity with implications for vaccine design. However, our data do not support the theory that genotype-3a viral clearance with therapy is associated with an enhanced antiviral T cell response. Paradoxically, a reduction in these responses may serve as a biomarker of IFN responsiveness.

Colloca S, Barnes E, Folgori A, Ammendola V, Capone S, Cirillo A, Siani L, Naddeo M et al. 2012. Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species. Sci Transl Med, 4 (115), pp. 115ra2. | Show Abstract | Read more

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.

Barnes E, Folgori A, Capone S, Swadling L, Aston S, Kurioka A, Meyer J, Huddart R et al. 2012. Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man. Sci Transl Med, 4 (115), pp. 115ra1. | Show Abstract | Read more

Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

O'Hara GA, Welten SP, Klenerman P, Arens R. 2012. Memory T cell inflation: understanding cause and effect. Trends Immunol, 33 (2), pp. 84-90. | Show Abstract | Read more

Typically, during viral infections, T cells encounter antigen, undergo proliferative expansion and ultimately contract into a pool of memory cells. However, after infection with cytomegalovirus, a ubiquitous β-herpesvirus, T cell populations specific for certain epitopes do not contract but instead are maintained and/or accumulate at high frequencies with a characteristic effector-memory phenotype. This feature has also been noted after other infections, for example, by parvoviruses. We discuss this so-called memory T cell inflation and the factors involved in this phenomenon. Also, we consider the potential therapeutic use of memory T cell inflation as a vaccine strategy and the associated implications for immune senescence.

Kang YH, Seigel B, Bengsch B, Fleming VM, Billerbeck E, Simmons R, Walker L, Willberg CB et al. 2012. CD161(+)CD4(+) T cells are enriched in the liver during chronic hepatitis and associated with co-secretion of IL-22 and IFN-γ. Front Immunol, 3 (NOV), pp. 346. | Show Abstract | Read more

Hepatitis C virus infection is a major cause of chronic liver disease. CD4(+) T cells play a key role in disease outcome. However, the critical functions and associated phenotypes of intrahepatic CD4(+) T cells are not well defined. We have previously shown that CD8(+) T cells expressing the C type lectin CD161 are highly enriched in the human liver, especially during chronic hepatitis. These cells are associated with a type 17 differentiation pattern and express cytokines including IL-17A, IL-22, and IFN-γ. We therefore analyzed expression of CD161 on CD4(+) T cells in blood and liver and addressed the relevant phenotype and functional capacity of these populations. We observed marked enrichment of CD161(+)CD4(+) T cells in the liver during chronic hepatitis such that they are the dominant subtype (mean 55% of CD4(+) T cells). IL-22 and IL-17 secreting CD4(+) T cells were readily found in the livers of HCV(+) and NASH donors, although not enriched compared to blood. There was, however, specific enrichment of a novel subset of IL-22/IFN-γ dual secretors (p = 0.02) compared to blood, a result reconfirmed with direct ex vivo analyses. These data indicate the dominance of CD161(+) expressing lymphocyte populations within the hepatic infiltrate, associated with a distinct cytokine profile. Given their documented roles as antiviral and hepatoprotective cytokines respectively, the impact of co-secretion of IFN-γ and IL-22 in the liver may be particularly significant.

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Oo YH, Banz V, Kavanagh D, Liaskou E, Withers DR, Humphreys E, Reynolds GM, Lee-Turner L et al. 2012. CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver Journal of Hepatology, 57 (5), pp. 1044-1051. | Show Abstract | Read more

Background & Aims: IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. Methods: The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. Results: IL-17+ T cells comprised 1-3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17+ cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. Conclusions: CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20. © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Humphreys IS, Von Delft A, Brown A, Hibbert L, Collier JD, Foster GR, Rahman M, Christian A, Klenerman P, Barnes E. 2012. HCV genotype-3a T cell immunity: Specificity, function and impact of therapy Gut, 61 (11), pp. 1589-1599. | Show Abstract | Read more

Background: Hepatitis C virus (HCV) genotype-3a infection is now the dominant strain in South Asia and the UK. Characteristic features include a favourable response to therapy; the reasons for this are unknown but may include distinct genotype-3a-specific T cell immunity. In contrast to genotype-1 infection, T cell immunity to this subtype is poorly defined. Objectives: The aims of the study were to (1) define the frequency, specificity and cross-reactivity of T cell immunity across the whole viral genome in genotype-3a infection and (2) assess the impact of interferon (IFN)-a/ribavirin on T cell immunity. Design: T cell responses in chronic and resolved HCV genotype-3a were analysed in comparison with genotype-1 infection (total n=85) using specific peptide panels in IFN-g ELISpot assays. T cell responses were followed longitudinally in a subset of genotype-3a infected patients receiving therapy. Responses were further defined by CD4 and CD8 subset analysis, sequencing of autologous virus and cross-reactivity of genotype-3a with genotype-1a/-1b antigens. Results: CD8 T cell responses commonly targeted the non-structural (NS) proteins in chronic genotype-3a infection whereas in genotype-1 infection CD4 responses targeting HCV core predominated (p=0.0183). Resolved infection was associated with CD4 T cells targeting NS proteins. Paradoxically, a sustained response to therapy was associated with a brisk decline in virus-specific and total lymphocyte counts that recovered after treatment. Conclusion: HCV genotype-3a exhibits a distinct T cell specificity with implications for vaccine design. However, our data do not support the theory that genotype-3a viral clearance with therapy is associated with an enhanced antiviral T cell response. Paradoxically, a reduction in these responses may serve as a biomarker of IFN responsiveness.

Klapa S, Schüler S, Pitann S, Klenerman P, Gross WL, Lamprecht P. 2012. Increased frequency of IL-7 and IL-15 receptor alpha chain (CD127, CD215) co-expressing CD4+ T cells in granulomatosis with polyangiitis (Wegener's) Clinical and Experimental Rheumatology, 30 (SUPPL. 70),

Klapa S, Schüler S, Pitann S, Klenerman P, Gross WL, Lamprecht P. 2012. Increased frequency of IL-7 and IL-15 receptor alpha chain (CD127, CD215) co-expressing CD4(+) T cells in granulomatosis with polyangiitis (Wegener's). Clin Exp Rheumatol, 30 (1 Suppl 70), pp. S171.

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Walker LJ, Kang YH, Smith MO, Tharmalingham H, Ramamurthy N, Fleming VM, Sahgal N, Leslie A et al. 2012. Human MAIT and CD8αα cells develop from a pool of type-17 precommitted CD8 <sup>+</sup> T cells Blood, 119 (2), pp. 422-433. | Show Abstract | Read more

Human mucosal associated invariant T (MAIT) CD8 + and Tc17 cells are important tissue-homing cell populations, characterized by high expression of CD161 ( ++) and type-17 differentiation, but their origins and relationships remain poorly defined. By transcriptional and functional analyses, we demonstrate that a pool of polyclonal, precommitted type-17CD161 ++CD8αβ + T cells exist in cord blood, from which a prominent MAIT cell(TCR Vα7.2 +) population emerges postnatally. During this expansion, CD8αα T cells appear exclusively within aCD161 ++CD8 +/MAIT subset, sharing cytokine production, chemokine-receptor expression, TCR-usage, and transcriptional profiles with their CD161 ++CD8αβ + counterparts. Our data demonstrate the origin and differentiation pathway of MAIT-cells from a naive type-17 precommitted CD161 ++CD8 +T-cell pool and the distinct phenotype and function of CD8αα cells in man. © 2012 by The American Society of Hematology.

Gangadharan B, Bapat M, Rossa J, Antrobus R, Chittenden D, Kampa B, Barnes E, Klenerman P, Dwek RA, Zitzmann N. 2012. Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients: a preliminary study. PLoS One, 7 (6), pp. e39603. | Show Abstract | Read more

BACKGROUND: Liver biopsy is the reference standard for assessing liver fibrosis and no reliable non-invasive diagnostic approach is available to discriminate between the intermediate stages of fibrosis. Therefore suitable serological biomarkers of liver fibrosis are urgently needed. We used proteomics to identify novel fibrosis biomarkers in hepatitis C patients with different degrees of liver fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: Proteins in plasma samples from healthy control individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using a proteomics technique: two dimensional gel electrophoresis (2-DE). This technique separated the proteins in plasma samples of control and cirrhotic patients and by visualizing the separated proteins we were able to identify proteins which were increasing or decreasing in hepatic cirrhosis. Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting. Forty four candidate biomarkers for hepatic fibrosis were identified of which 20 were novel biomarkers of liver fibrosis. Western blot validation of all candidate markers using plasma samples from patients across all Ishak fibrosis scores showed that the markers which changed with increasing fibrosis most consistently included lipid transfer inhibitor protein, complement C3d, corticosteroid-binding globulin, apolipoprotein J and apolipoprotein L1. These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect. These markers will be further validated using a large clinical cohort. CONCLUSIONS/SIGNIFICANCE: This study identifies 20 novel fibrosis biomarker candidates. The proteins identified may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies.

Klapa S, Schueler S, Pitann S, Klenerman P, Gross WL, Lamprecht P. 2012. Increased frequency of IL-7 and IL-15 receptor alpha chain (CD127, CD215) co-expressing CD4+T cells in granulomatosis with polyangiitis (Wegener's) CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, 30 (1), pp. S171-S171.

Mitchell JE, Chetty S, Govender P, Pillay M, Jaggernath M, Kasmar A, Ndung'u T, Klenerman P, Walker BD, Kasprowicz VO. 2012. Prospective monitoring reveals dynamic levels of T cell immunity to Mycobacterium tuberculosis in HIV infected individuals. PLoS One, 7 (6), pp. e37920. | Show Abstract | Read more

Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot) on 163 HIV-infected individuals living in a TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3-21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from -150 to +153 spot-forming cells (SFC)/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) (MIG: r=0.6527, p=0.0114; IP-10: r=0.6967, p=0.0056; IP-10+MIG: r=0.7055, p=0.0048). During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.

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Scopus

O'Hara GA, Welten SPM, Klenerman P, Arens R. 2012. Memory T cell inflation: Understanding cause and effect Trends in Immunology, 33 (2), pp. 84-90. | Show Abstract | Read more

Typically, during viral infections, T cells encounter antigen, undergo proliferative expansion and ultimately contract into a pool of memory cells. However, after infection with cytomegalovirus, a ubiquitous β-herpesvirus, T cell populations specific for certain epitopes do not contract but instead are maintained and/or accumulate at high frequencies with a characteristic effector-memory phenotype. This feature has also been noted after other infections, for example, by parvoviruses. We discuss this so-called memory T cell inflation and the factors involved in this phenomenon. Also, we consider the potential therapeutic use of memory T cell inflation as a vaccine strategy and the associated implications for immune senescence. © 2011 Elsevier Ltd.

Xue L, Barrow A, Fleming VM, Hunter MG, Ogg G, Klenerman P, Pettipher R. 2012. Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine production in response to prostaglandin D2. J Immunol, 188 (2), pp. 694-702. | Show Abstract | Read more

PGD(2) exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD(2). PGD(2) induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D(4) and E(4) (LTE(4)) also stimulated the cytokine production but were much less active than PGD(2). However, when combined with PGD(2), cysLTs caused a greater than additive enhancement of the response, with LTE(4) being most effective in activating Th2 cells. LTE(4) enhanced calcium mobilization in response to PGD(2) in Th2 cells without affecting endogenous PGD(2) production or CRTH2 receptor expression. The effect of LTE(4) was inhibited by montelukast but not by the P2Y(12) antagonist methylthioadenosine 5'-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by MK886, an inhibitor of 5-lipoxygenase-activating protein, reduced the response of Th2 cells to the levels produced by PGD(2) alone. These findings reveal that cysLTs, in particular LTE(4), have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor.

Klenerman P, Gupta PK. 2012. Hepatitis C virus: current concepts and future challenges. QJM, 105 (1), pp. 29-32. | Read more

Klenerman P. 2012. Viral infection and immunity: balancing protection and pathology. QJM, 105 (1), pp. 103-104. | Read more

Walker LJ, Kang YH, Smith MO, Tharmalingham H, Ramamurthy N, Fleming VM, Sahgal N, Leslie A et al. 2012. Human MAIT and CD8αα cells develop from a pool of type-17 precommitted CD8+ T cells. Blood, 119 (2), pp. 422-433. | Show Abstract | Read more

Human mucosal associated invariant T (MAIT) CD8(+) and Tc17 cells are important tissue-homing cell populations, characterized by high expression of CD161 ((++)) and type-17 differentiation, but their origins and relationships remain poorly defined. By transcriptional and functional analyses, we demonstrate that a pool of polyclonal, precommitted type-17 CD161(++)CD8αβ(+) T cells exist in cord blood, from which a prominent MAIT cell (TCR Vα7.2(+)) population emerges post-natally. During this expansion, CD8αα T cells appear exclusively within a CD161(++)CD8(+)/MAIT subset, sharing cytokine production, chemokine-receptor expression, TCR-usage, and transcriptional profiles with their CD161(++)CD8αβ(+) counterparts. Our data demonstrate the origin and differentiation pathway of MAIT-cells from a naive type-17 precommitted CD161(++)CD8(+) T-cell pool and the distinct phenotype and function of CD8αα cells in man.

Li T, Chen Z, Zeng J, Zhang J, Wang W, Zhang L, Zheng X, Shuai L, Klenerman P, Allain JP, Li C. 2011. Impact of host responses on control of hepatitis C virus infection in Chinese blood donors. Biochem Biophys Res Commun, 415 (3), pp. 503-508. | Show Abstract | Read more

A study was undertaken to explore the molecular mechanisms underlying control of HCV infection in blood donors in China. Factors including clinical information, anti-HCV reactivity (S/CO), IFN-α and IFN-γ, viral loads and genotypes were correlated with 160 index plasma samples at three statuses of 45 recovered, 76 chronic or 39 false positive anti-HCV reactive blood donors. The spontaneous recovery rate was 37.2%. Viral loads of 76 HCV plasmas ranged between 59.8 IU/ml and 2.43 × 10(6)IU/ml (median 3.67 × 10(4)IU/ml). Genotypes 1, 2, 3 and 6 of 63 HCV strains were identified phylogenetically. Recovered donors were significantly younger (p=0.002) and had lower level IFN-γ (p=0.001) than chronically HCV infected donors. Circulating levels of IFN-α and IFN-γ were higher in those with low viral load and were low in middle or high viral load samples. The ratio of IFN-α to IFN-γ (IFN-α/γ) was significantly positively correlated with viral load (p=0.037), and viral load was inversely correlated with IFN-γ in chronic HCV infection regardless of genotype. The study revealed clearly different relationships between IFN-α and IFN-γ in relation to viral load in HCV. A novel measure of IFN-α/γ ratio could be a new approach to evaluate long term outcome of HCV infection.

Wills M, Akbar A, Beswick M, Bosch JA, Caruso C, Colonna-Romano G, Dutta A, Franceschi C et al. 2011. Report from the second cytomegalovirus and immunosenescence workshop. Immun Ageing, 8 (1), pp. 10. | Show Abstract | Read more

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.

Marrinan E, Walker L, Barnes E, Klenerman P. 2011. CD8B LOW T CELLS ARE A PROMINENT, FUNCTIONALLY DISTINCT POPULATION IN CHRONIC HEPATITIS B INFECTION GUT, 60 (Suppl 2), pp. A55-+. | Read more

Rajoriya N, Willberg C, Seigal B, Kang Y, Phillips-Hughes J, Collier J, Barnes E, Thimme R, Klenerman P. 2011. CD161+gamma-delta T CELLS: DEFINING THEIR ROLE IN PATIENTS WITH AND WITHOUT CHRONIC HEPATITIS C GUT, 60 (Suppl 2), pp. A43-A43. | Read more

Klenerman P, Thimme R. 2012. T cell responses in hepatitis C: the good, the bad and the unconventional. Gut, 61 (8), pp. 1226-1234. | Show Abstract | Read more

Over recent years, it has become increasingly accepted that virus-specific CD4+ and CD8+ T cell responses play a major role in outcome and pathogenesis of hepatitis C virus (HCV) infection. Indeed, while the emergence of strong and multispecific T cell responses may correlate with spontaneous viral clearance, the virus has developed several mechanisms to avoid T cell control in the majority of acutely HCV-infected patients that subsequently develop persistent HCV infection. In this review, we will discuss the current knowledge about the role of cellular immune responses in HCV infection. Specifically, we will emphasise recent new insights into the effector functions of T cells, possible mechanisms of their failure and the host-virus interactions occurring at the site of the disease, the liver.

Thomson EC, Smith JA, Klenerman P. 2011. The natural history of early hepatitis C virus evolution; lessons from a global outbreak in human immunodeficiency virus-1-infected individuals. J Gen Virol, 92 (Pt 10), pp. 2227-2236. | Show Abstract | Read more

New insights into the early viral evolution and cellular immune response during acute hepatitis C virus (HCV) infection are being gained following a global outbreak in human immunodeficiency virus-1 (HIV)-positive men who have sex with men. Cross-sectional and longitudinal sequence analysis at both the population and individual level have facilitated tracking of the HCV epidemic across the world and enabled the development of tests of viral diversity in individual patients in order to predict spontaneous clearance of HCV and response to treatment. Immunological studies in HIV-positive cohorts have highlighted the role of the CD4+ T-cell response in the control of early HCV infection and will increase the opportunity for the identification of protective epitopes that could be used in future vaccine development.

Fitzmaurice K, Petrovic D, Ramamurthy N, Simmons R, Merani S, Gaudieri S, Sims S, Dempsey E et al. 2011. Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection. Gut, 60 (11), pp. 1563-1571. | Show Abstract | Read more

BACKGROUND AND AIMS: CD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response. METHODS: A sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants. RESULTS: A strong 'HLA footprint' in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03-positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A. CONCLUSIONS: It is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.

Huang GJ, Smith AL, Gray DH, Cosgrove C, Singer BH, Edwards A, Sim S, Parent JM et al. 2011. Correction: A Genetic and Functional Relationship between T Cells and Cellular Proliferation in the Adult Hippocampus. PLoS Biol, 9 (5), | Show Abstract | Read more

[This corrects the article on p. e1000561 in vol. 8.].

Nebbia G, Mattes FM, Smith C, Hainsworth E, Kopycinski J, Burroughs A, Griffiths PD, Klenerman P, Emery VC. 2011. Polyfunctional Cytomegalovirus-Specific CD4(+) and pp65 CD8(+) T Cells Protect Against High-Level Replication After Liver Transplantation. (vol 8, pg 2590, 2008) AMERICAN JOURNAL OF TRANSPLANTATION, 11 (5), pp. 1107-1107.

Mattes FM, Vargas A, Kopycinski J, Hainsworth EG, Sweny P, Nebbia G, Bazeos A, Lowdell M et al. 2011. Functional Impairment of Cytomegalovirus Specific CD8 T Cells Predicts High-Level Replication After Renal Transplantation. American Journal of Transplantation (vol 8, pg 990, 2008) AMERICAN JOURNAL OF TRANSPLANTATION, 11 (5), pp. 1107-1107.

Halliday J, Klenerman P, Barnes E. 2011. Vaccination for hepatitis C virus: closing in on an evasive target. Expert Rev Vaccines, 10 (5), pp. 659-672. | Show Abstract | Read more

Hepatitis C virus (HCV) infects more than 170 million people globally and is a leading cause of liver cirrhosis, transplantation and hepatocellular carcinoma. Current gold-standard therapy often fails, has significant side effects in many cases and is expensive. No vaccine is currently available. The fact that a significant proportion of infected people spontaneously control HCV infection in the setting of an appropriate immune response suggests that a vaccine for HCV is a realistic goal. A comparative analysis of infected people with distinct clinical outcomes has enabled the characterization of many important innate and adaptive immune processes associated with viral control. It is clear that a successful HCV vaccine will need to exploit and enhance these natural immune defense mechanisms. New HCV vaccine approaches, including peptide, recombinant protein, DNA and vector-based vaccines, have recently reached Phase I/II human clinical trials. Some of these technologies have generated robust antiviral immunity in healthy volunteers and infected patients. The challenge now is to move forward into larger at-risk or infected populations to truly test efficacy.

Kasprowicz V, Kang Y-H, Lucas M, zur Wiesch JS, Kuntzen T, Fleming V, Nolan BE, Longworth S et al. 2011. Hepatitis C Virus (HCV) Sequence Variation Induces an HCV-Specific T-Cell Phenotype Analogous to Spontaneous Resolution (vol 84, pg 1656, 2010) JOURNAL OF VIROLOGY, 85 (9), pp. 4636-4636. | Read more

Matthews PC, Leslie AJ, Katzourakis A, Crawford H, Payne R, Prendergast A, Power K, Kelleher AD et al. 2011. HLA Footprints on Human Immunodeficiency Virus Type 1 Are Associated with Interclade Polymorphisms and Intraclade Phylogenetic Clustering (vol 83, pg 4605, 2009) JOURNAL OF VIROLOGY, 85 (9), pp. 4635-4635. | Read more

Neumann-Haefelin C, Frick DN, Wang JJ, Pybus OG, Salloum S, Narula GS, Eckart A, Biezynski A et al. 2011. Analysis of the Evolutionary Forces in an Immunodominant CD8 Epitope in Hepatitis C Virus at a Population Level (vol 82, pg 3438, 2008) JOURNAL OF VIROLOGY, 85 (9), pp. 4634-4634. | Read more

Kasprowicz V, zur Wiesch JS, Kuntzen T, Nolan BE, Longworth S, Berical A, Blum J, McMahon C et al. 2011. High Level of PD-1 Expression on Hepatitis C Virus (HCV)-Specific CD8(+) and CD4(+) T Cells during Acute HCV Infection, Irrespective of Clinical Outcome (vol 82, pg 3154, 2008) JOURNAL OF VIROLOGY, 85 (9), pp. 4633-4633. | Read more

Simmons R, Sharp C, Sims S, Kloverpris H, Goulder P, Simmonds P, Bowness P, Klenerman P. 2011. High frequency, sustained T cell responses to PARV4 suggest viral persistence in vivo. J Infect Dis, 203 (10), pp. 1378-1387. | Show Abstract | Read more

BACKGROUND: Parvovirus 4 (PARV4) is a recently identified human virus that has been found in livers of patients infected with hepatitis C virus (HCV) and in bone marrow of individuals infected with human immunodeficiency virus (HIV). T cells are important in controlling viruses but may also contribute to disease pathogenesis. The interaction of PARV4 with the cellular immune system has not been described. Consequently, we investigated whether T cell responses to PARV4 could be detected in individuals exposed to blood-borne viruses. METHODS: Interferon γ (IFN-γ) enzyme-linked immunospot assay, intracellular cytokine staining, and a tetrameric HLA-A*0201-peptide complex were used to define the lymphocyte populations responding to PARV4 NS peptides in 88 HCV-positive and 13 HIV-positive individuals. Antibody responses were tested using a recently developed PARV4 enzyme-linked immunosorbent assay. RESULTS: High-frequency T cell responses against multiple PARV4 NS peptides and antibodies were observed in 26% of individuals. Typical responses to the NS pools were >1000 spot-forming units per million peripheral blood mononuclear cells. CONCLUSIONS: PARV4 infection is common in individuals exposed to blood-borne viruses and elicits strong T cell responses, a feature typically associated with persistent, contained infections such as cytomegalovirus. Persistence of PARV4 viral antigen in tissue in HCV-positive and HIV-positive individuals and/or the associated activated antiviral T cell response may contribute to disease pathogenesis.

Marashi SM, Raeiszadeh M, Workman S, Rahbar A, Soderberg-Naucler C, Klenerman P, Chee R, Webster AD, Milne RS, Emery VC. 2011. Inflammation in common variable immunodeficiency is associated with a distinct CD8(+) response to cytomegalovirus. J Allergy Clin Immunol, 127 (6), pp. 1385-93.e4. | Show Abstract | Read more

BACKGROUND: Common variable immunodeficiency is the most common primary immunodeficiency. A subset of patients has debilitating inflammatory complications. OBJECTIVES: We investigated the role of cytomegalovirus (CMV), and the T-cell response targeted at this virus, in this inflammatory disease. METHODS: Phenotypic and functional assays were used to profile CMV-specific T cells in patients with common variable immunodeficiency with and without inflammatory complications. Highly sensitive immunohistochemistry was used to detect CMV antigens at sites of inflammation. RESULTS: Cytomegalovirus was significantly associated with inflammatory disease, which occurred in 31 of 43 (72%) virus-exposed patients and 8 of 31 (26%) naive patients (P = .0001). CMV pp65-NLVPMVATV epitope-specific CD8(+) T-cell frequencies were significantly elevated in inflammatory patients, but these cells did not show evidence of exhaustion, with low levels of programmed death-1 and high T-cell receptor avidity. Rather, they showed features consistent with high in vivo functionality and proliferative activity including reduced levels of the anti-inflammatory marker CD73 (1.67% of NLV(+) cells were CD73(+) vs 42.01% in noninflammatory patients; P = .004) and increased Ki-67 expression (37% vs 2% in noninflammatory patients; P < .0001). In vitro, the CMV-specific T cells showed high antigen-specific proliferative potential compared with cells from noninflammatory patients. By using sensitive immunohistochemistry, we detected for the first time viral antigen at the sites of inflammation, indicative of active viral replication. CONCLUSION: Our data strongly support a direct role for CMV and a hyperreactive CMV-specific immune response in the debilitating chronic inflammatory complications of common variable immunodeficiency.

Thomson E, Main J, Karayiannis P, Klenerman P. 2011. Intravenous methamphetamine use is associated with lower sustained virological response rates in HIV-positive men who have sex with men infected with acute hepatitis C HIV MEDICINE, 12 pp. 26-26.

Todryk SM, Pathan AA, Keating S, Porter DW, Berthoud T, Thompson F, Klenerman P, Hill AVS. 2011. The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming (vol 128, pg 83, 2009) IMMUNOLOGY, 132 (4), pp. 589-589. | Read more

Gangadharan B, Antrobus R, Chittenden D, Rossa J, Bapat M, Klenerman P, Barnes E, Dwek RA, Zitzmann N. 2011. New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients. J Proteome Res, 10 (5), pp. 2643-2650. | Show Abstract | Read more

Despite many shortcomings, liver biopsy is regarded as the gold standard for assessing liver fibrosis. A less invasive and equally or more reliable approach would constitute a major advancement in the field. Proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood. A major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins, which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features. To overcome this problem, we used two-dimensional gel electrophoresis (2-DE) over a narrow pH 3-5.6 range since this lies outside the range of highly abundant albumin, transferrin and immunoglobulins. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in hepatitis C induced liver cirrhosis. Using the pH 3-5.6 range for 2-DE, we achieved improved representation of low abundance features and enhanced separation. We found in-solution isoelectric focusing to be beneficial for analyzing basic, high molecular weight proteins. Using this method, the beta chains of both complement C3 and C4 were found to decrease in serum from hepatitis C patients with cirrhosis, a change not observed previously by 2-DE. We present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis.

Barnes E, Folgori A, Capone S, Aston S, Meyer J, Gantlett K, Smith KE, Huddart R et al. 2011. PHASE I TRIAL OF A HIGHLY IMMUNOGENIC AND DURABLE T-CELL VACCINE FOR HEPATITIS C VIRUS BASED ON NOVEL, RARE, ADENOVIRAL VECTORS JOURNAL OF HEPATOLOGY, 54 pp. S450-S451.

Kelly C, Folgori A, Capone S, Stafford E, O'Donnell D, Gantlett K, Collier J, Brown AC et al. 2011. A THERAPEUTIC VACCINE FOR HCV BASED ON NOVEL, RARE, ADENOVIRAL VECTORS JOURNAL OF HEPATOLOGY, 54 pp. S29-S29.

Bucci C, Rahman M, Humphreys I, Brown AC, Lucas M, Pfafferott K, Collier J, O'Donnell D et al. 2011. IL28B GENETIC STATUS IN ASSOCIATION WITH HCV SPECIFIC T-CELL IMMUNITY JOURNAL OF HEPATOLOGY, 54 pp. S38-S38.

Kasprowicz V, Ward SM, Turner A, Grammatikos A, Nolan BE, Lewis-Ximenez L, Sharp C, Woodruff J et al. 2011. Defining the directionality and quality of influenza virus-specific CD8(+) T cell cross-reactivity in individuals infected with hepatitis C virus (vol 118, pg 1143, 2008) JOURNAL OF CLINICAL INVESTIGATION, 121 (3), pp. 1223-1223. | Read more

di Iulio J, Ciuffi A, Fitzmaurice K, Kelleher D, Rotger M, Fellay J, Martinez R, Pulit S et al. 2011. Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance. Hepatology, 53 (5), pp. 1446-1454. | Show Abstract | Read more

UNLABELLED: The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. CONCLUSION: We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Arens R, Loewendorf A, Redeker A, Sierro S, Boon L, Klenerman P, Benedict CA, Schoenberger SP. 2011. Differential B7-CD28 costimulatory requirements for stable and inflationary mouse cytomegalovirus-specific memory CD8 T cell populations. J Immunol, 186 (7), pp. 3874-3881. | Show Abstract | Read more

CMV establishes a lifelong persistent infection, and viral immune-modulating strategies are important in facilitating this. A particularly diverse CD8 T cell response develops as a result of this host-virus détente, with the CMV-specific memory T cell pool displaying unique functions and phenotypes. To gain insight into the factors that regulate CMV-specific CD8 T cell responses, we examined the influence of the B7-CD28 costimulatory pathway on magnitude, kinetics, and phenotype. Initial expansion of mouse CMV-specific CD8 T cells that establish stable memory pools was severely lower in mice lacking B7-CD28 signaling, and the resulting memory levels also remained reduced during persistent/latent infection. In contrast, expansion of CD8 T cells that undergo memory inflation during chronic infection was less affected in the absence of B7-CD28 costimulatory signals, eventually reaching the levels seen in wild-type mice at later times. Regardless of their differential requirements for B7-CD28 signals, both stable and inflationary memory T cell populations showed normal cytotoxic capacity. These results reveal that B7-CD28 costimulation differentially regulates the magnitude and kinetics of the multifaceted CD8 T cell response that develops during CMV infection.

Klenerman P, Webb A, McPherson T, Brennan N, Petrou A, Prassas E, Sullivan P, Adams E et al. 2011. Proceedings of Research in Clinical Practice 2010: Research in Clinical Practice was held at the Academic Centre of the John Radcliffe Hospital, Oxford, on Wednesday, 10 November 2010. QJM, 104 (4), pp. 345-364. | Read more

Kelly C, Klenerman P, Barnes E. 2011. Interferon lambdas: the next cytokine storm. Gut, 60 (9), pp. 1284-1293. | Show Abstract | Read more

For two decades the scientific community has sought to understand why some people clear hepatitis C virus (HCV) and others do not. Recently, several large genome-wide association studies have identified single nucleotide polymorphisms (SNPs) linked to interferon lambda 3 (IFNλ3) that are associated with the spontaneous resolution and successful treatment of HCV infection. These observations are generating intense research activity; the hope is that IFNλ3 genetic variants may serve as important predictive biomarkers of treatment outcome and offer new insights into the biological pathways involved in viral control. A pharmacogenomic treatment approach for HCV can now be envisaged, with the incorporation of host genetic variants into a predictive treatment algorithm with other factors. The SNPs associated with the clinical outcome of HCV infection are located some distance from the IFNλ3 gene itself, and causal genetic variants have yet to be clearly defined. Locating these causal variants, mapping in detail the IFNλ3 signalling pathways and determining the downstream genetic signature so induced will clarify the role of IFNλ3 in the pathogenesis of HCV. Clinical studies assessing safety and efficacy in the treatment of HCV with exogenous IFNλ3 are currently underway. Early results suggest that IFNλ3 treatment inhibits HCV replication and is associated with a limited side effect profile. However, hepatotoxicity in both healthy volunteers and HCV-infected patients has been described. This review discusses the genetic studies that link IFNλ3 to both the spontaneous resolution and treatment-induced clearance of HCV and the potential impact of this in clinical practice, the biology of IFNλ3 as currently understood and how this may impact on HCV infection, and describes the early studies that assess the role of this cytokine in the treatment of patients with HCV.

Fergusson JR, Fleming VM, Klenerman P. 2011. CD161-expressing human T cells. Front Immunol, 2 (AUG), pp. 36. | Show Abstract | Read more

Expression of the Natural Killer cell receptor CD161 has recently been identified on a subset of T cells, including both CD4+ T helper and CD8+ T cells. Expression of this molecule within the adult circulation is restricted to those T cells with a memory phenotype. However, the distinct properties of these T cell populations is yet to be fully determined, although expression of CD161 has been related to the secretion of interleukin-17, and therefore to a type 17 phenotype. Recent studies have aimed to determine both the origin of these cells and the significance of CD161 expression as either a marker of specific cell types or as an effector and regulator of lymphocyte function, and hence to characterize the role of these CD161+ cells within a variety of human diseases in which they have been implicated.

Li T, Chen Z, Zeng J, Zhang J, Wang W, Zhang L, Zheng X, Shuai L, Klenerman P, Allain JP, Li C. 2011. Impact of host responses on control of hepatitis C virus infection in Chinese blood donors Biochemical and Biophysical Research Communications, 415 (3), pp. 503-508. | Show Abstract | Read more

A study was undertaken to explore the molecular mechanisms underlying control of HCV infection in blood donors in China. Factors including clinical information, anti-HCV reactivity (S/CO), IFN-α and IFN-γ, viral loads and genotypes were correlated with 160 index plasma samples at three statuses of 45 recovered, 76 chronic or 39 false positive anti-HCV reactive blood donors. The spontaneous recovery rate was 37.2%. Viral loads of 76 HCV plasmas ranged between 59.8IU/ml and 2.43×10 6IU/ml (median 3.67×10 4IU/ml). Genotypes 1, 2, 3 and 6 of 63 HCV strains were identified phylogenetically. Recovered donors were significantly younger (p=0.002) and had lower level IFN-γ (p=0.001) than chronically HCV infected donors. Circulating levels of IFN-α and IFN-γ were higher in those with low viral load and were low in middle or high viral load samples. The ratio of IFN-α to IFN-γ (IFN-α/γ) was significantly positively correlated with viral load (p=0.037), and viral load was inversely correlated with IFN-γ in chronic HCV infection regardless of genotype. The study revealed clearly different relationships between IFN-α and IFN-γ in relation to viral load in HCV. A novel measure of IFN-α/γ ratio could be a new approach to evaluate long term outcome of HCV infection. © 2011 Elsevier Inc.

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Fitzmaurice K, Petrovic D, Ramamurthy N, Simmons R, Merani S, Gaudieri S, Sims S, Dempsey E et al. 2011. Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection Gut, 60 (11), pp. 1563-1571. | Show Abstract | Read more

Background and aims: CD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response. Methods: A sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants. Results: A strong 'HLA footprint' in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03- positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A. Conclusions: It is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.

Thomson EC, Smith JA, Klenerman P. 2011. The natural history of early hepatitis C virus evolution; Lessons from a global outbreak in human immunodeficiency virus-1-infected individuals Journal of General Virology, 92 (10), pp. 2227-2236. | Show Abstract | Read more

New insights into the early viral evolution and cellular immune response during acute hepatitis C virus (HCV) infection are being gained following a global outbreak in human immunodeficiency virus-1 (HIV)-positive men who have sex with men. Cross-sectional and longitudinal sequence analysis at both the population and individual level have facilitated tracking of the HCV epidemic across the world and enabled the development of tests of viral diversity in individual patients in order to predict spontaneous clearance of HCV and response to treatment. Immunological studies in HIV-positive cohorts have highlighted the role of the CD4 + T-cell response in the control of early HCV infection and will increase the opportunity for the identification of protective epitopes that could be used in future vaccine development. © 2011 SGM.

Klenerman P, Thimme R. 2011. T cell responses in hepatitis C: The good, the bad and the unconventional Gut,

Kasprowicz VO, Mitchell JE, Chetty S, Govender P, Huang KHG, Fletcher HA, Webster DP, Brown S et al. 2011. A molecular assay for sensitive detection of pathogen-specific T-cells PLoS ONE, 6 (8), | Show Abstract | Read more

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P&lt;0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings. © 2011 Kasprowicz et al.

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Kelly C, Klenerman P, Barnes E. 2011. Interferon lambdas: The next cytokine storm Gut, 60 (9), pp. 1284-1293. | Show Abstract | Read more

For two decades the scientific community has sought to understand why some people clear hepatitis C virus (HCV) and others do not. Recently, several large genome-wide association studies have identified single nucleotide polymorphisms (SNPs) linked to interferon lambda 3 (IFNλ3) that are associated with the spontaneous resolution and successful treatment of HCV infection. These observations are generating intense research activity; the hope is that IFNλ3 genetic variants may serve as important predictive biomarkers of treatment outcome and offer new insights into the biological pathways involved in viral control. A pharmacogenomic treatment approach for HCV can now be envisaged, with the incorporation of host genetic variants into a predictive treatment algorithm with other factors. The SNPs associated with the clinical outcome of HCV infection are located some distance from the IFNλ3 gene itself, and causal genetic variants have yet to be clearly defined. Locating these causal variants, mapping in detail the IFNλ3 signalling pathways and determining the downstream genetic signature so induced will clarify the role of IFNλ3 in the pathogenesis of HCV. Clinical studies assessing safety and efficacy in the treatment of HCV with exogenous IFNλ3 are currently underway. Early results suggest that IFNλ3 treatment inhibits HCV replication and is associated with a limited side effect profile. However, hepatotoxicity in both healthy volunteers and HCV-infected patients has been described. This review discusses the genetic studies that link IFNλ3 to both the spontaneous resolution and treatment-induced clearance of HCV and the potential impact of this in clinical practice, the biology of IFNλ3 as currently understood and how this may impact on HCV infection, and describes the early studies that assess the role of this cytokine in the treatment of patients with HCV.

Kasprowicz VO, Mitchell JE, Chetty S, Govender P, Huang KH, Fletcher HA, Webster DP, Brown S et al. 2011. A molecular assay for sensitive detection of pathogen-specific T-cells. PLoS One, 6 (8), pp. e20606. | Show Abstract | Read more

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters--monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

Marashi SM, Raeiszadeh M, Workman S, Rahbar A, Soderberg-Naucler C, Klenerman P, Chee R, Webster AD, Milne RSB, Emery VC. 2011. Inflammation in common variable immunodeficiency is associated with a distinct CD8+ response to cytomegalovirus Journal of Allergy and Clinical Immunology, 127 (6), | Show Abstract | Read more

Background: Common variable immunodeficiency is the most common primary immunodeficiency. A subset of patients has debilitating inflammatory complications. Objectives: We investigated the role of cytomegalovirus (CMV), and the T-cell response targeted at this virus, in this inflammatory disease. Methods: Phenotypic and functional assays were used to profile CMV-specific T cells in patients with common variable immunodeficiency with and without inflammatory complications. Highly sensitive immunohistochemistry was used to detect CMV antigens at sites of inflammation. Results: Cytomegalovirus was significantly associated with inflammatory disease, which occurred in 31 of 43 (72%) virus-exposed patients and 8 of 31 (26%) naive patients (P = .0001). CMV pp65-NLVPMVATV epitope-specific CD8+ T-cell frequencies were significantly elevated in inflammatory patients, but these cells did not show evidence of exhaustion, with low levels of programmed death-1 and high T-cell receptor avidity. Rather, they showed features consistent with high in vivo functionality and proliferative activity including reduced levels of the anti-inflammatory marker CD73 (1.67% of NLV+ cells were CD73 + vs 42.01% in noninflammatory patients; P = .004) and increased Ki-67 expression (37% vs 2% in noninflammatory patients; P < .0001). In vitro, the CMV-specific T cells showed high antigen-specific proliferative potential compared with cells from noninflammatory patients. By using sensitive immunohistochemistry, we detected for the first time viral antigen at the sites of inflammation, indicative of active viral replication. Conclusion: Our data strongly support a direct role for CMV and a hyperreactive CMV-specific immune response in the debilitating chronic inflammatory complications of common variable immunodeficiency. © 2011 American Academy of Allergy, Asthma & Immunology.

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Thomson EC, Fleming VM, Main J, Klenerman P, Weber J, Eliahoo J, Smith J, McClure MO, Karayiannis P. 2011. Predicting spontaneous clearance of acute hepatitis C virus in a large cohort of HIV-1-infected men Gut, 60 (6), pp. 837-845. | Show Abstract | Read more

Objective: An epidemic of acute hepatitis C virus (HCV) infection in HIV-positive men-who-have-sex-with-men (MSM) is emerging in Europe, Australia and the USA. The aim of this study was to characterise the natural history of primary HCV in this setting and to assess host and viral factors which predict spontaneous clearance. Methods This prospective longitudinal cohort study was carried out in 112 HIV-positive patients who were followed in a single centre (the St Mary's Acute HCV Cohort). Plasma and peripheral blood mononuclear cells (PBMCs) were obtained at monthly intervals for 3 months and at 3-monthly intervals thereafter for a median of 45 months (IQR=29-69 months). The primary end point was spontaneous clearance of HCV. Cox regression was used to assess the impact of clinical and virological variables on outcome, including liver function, CD4 count, rate of HCV RNA decline, T cell response and clonal sequence evolution within the HCV E2 envelope gene. Results: 15% of patients cleared HCV spontaneously, while 85% progressed towards chronicity. The latter group included a significant proportion of 'fluctuating' progressors (37.5%), in whom a fall followed by a rise (>1 log10) in viraemia was observed. This was associated with superinfection with new HCV strains and partially effective T cell responses. Spontaneous clearance was strongly associated with a 2.2 log10 viral load drop within 100 days of infection (HR=1.78; p<0.0001), elevated bilirubin (≥40 μmol/l; HR=5.04; p=0.006), elevated alanine aminotransferase (ALT; ≥1000 IU/ml; HR=2.62; p=0.048) and baseline CD4 count ≥650×106/l (HR=2.66; p=0.045), and only occurred in patients with genotype 1 infection. Evolution to spontaneous clearance occurred in patients with low viral diversity in the presence of an early multispecific T cell response. Conclusions: Spontaneous clearance of acute HCV in HIV-positive men can be predicted by a rapid decline in viral load, high CD4 count, elevated bilirubin and ALT, and is associated with low viral diversity and strong T cell responses.

Mattes FM, Vargas A, Kopycinski J, Hainsworth EG, Sweny P, Nebbia G, Bazeos A, Lowdell M et al. 2011. Erratum American Journal of Transplantation, 11 (5), pp. 1107-1107. | Read more

Neumann-Haefelin C, Frick DN, Wang JJ, Pybus OG, Salloum S, Narula GS, Eckart A, Biezynski A et al. 2011. Analysis of the evolutionary forces in an immunodominant CD8 epitope in hepatitis C virus at a population level Journal of Virology, 85 (9), pp. 4634. | Read more

Kasprowicz V, Schulze zur Wiesch J, Kuntzen T, Nolan BE, Longworth S, Berical A, Blum J, McMahon C, Reyor LL, Elias N. 2011. High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8<sup>+</sup> and CD4<sup>+</sup> T cells during acute HCV infection, irrespective of clinical outcome Journal of Virology, 85 (9), pp. 4633. | Read more

Kasprowicz V, Kang YH, Lucas M, Schulze zur Wiesch J, Kuntzen T, Fleming V, Nolan BE, Longworth S et al. 2011. Hepatitis C virus (HCV) sequence variation induces an HCV-specific T-cell phenotype analogous to spontaneous resolution Journal of Virology, 85 (9), pp. 4636. | Read more

Huang KH, Goedhals D, Carlson JM, Brockman MA, Mishra S, Brumme ZL, Hickling S, Tang CS et al. 2011. Progression to AIDS in South Africa is associated with both reverting and compensatory viral mutations. PLoS One, 6 (4), pp. e19018. | Show Abstract | Read more

We lack the understanding of why HIV-infected individuals in South Africa progress to AIDS. We hypothesised that in end-stage disease there is a shifting dynamic between T cell imposed immunity and viral immune escape, which, through both compensatory and reverting viral mutations, results in increased viral fitness, elevated plasma viral loads and disease progression. We explored how T cell responses, viral adaptation and viral fitness inter-relate in South African cohorts recruited from Bloemfontein, the Free State (n = 278) and Durban, KwaZulu-Natal (n = 775). Immune responses were measured by γ-interferon ELISPOT assays. HLA-associated viral polymorphisms were determined using phylogenetically corrected techniques, and viral replication capacity (VRC) was measured by comparing the growth rate of gag-protease recombinant viruses against recombinant NL4-3 viruses. We report that in advanced disease (CD4 counts <100 cells/µl), T cell responses narrow, with a relative decline in Gag-directed responses (p<0.0001). This is associated with preserved selection pressure at specific viral amino acids (e.g., the T242N polymorphism within the HLA-B*57/5801 restricted TW10 epitope), but with reversion at other sites (e.g., the T186S polymorphism within the HLA-B*8101 restricted TL9 epitope), most notably in Gag and suggestive of "immune relaxation". The median VRC from patients with CD4 counts <100 cells/µl was higher than from patients with CD4 counts ≥ 500 cells/µl (91.15% versus 85.19%, p = 0.0004), potentially explaining the rise in viral load associated with disease progression. Mutations at HIV Gag T186S and T242N reduced VRC, however, in advanced disease only the T242N mutants demonstrated increasing VRC, and were associated with compensatory mutations (p = 0.013). These data provide novel insights into the mechanisms of HIV disease progression in South Africa. Restoration of fitness correlates with loss of viral control in late disease, with evidence for both preserved and relaxed selection pressure across the HIV genome. Interventions that maintain viral fitness costs could potentially slow progression.

Kasprowicz V, Ward SM, Turner A, Grammatikos A, Nolan BE, Lewis-Ximenez L, Sharp C, Woodruff J et al. 2011. Defining the directionality and quality of influenza virus-specific CD8+ T cell cross-reactivity in individuals infected with hepatitis C virus (Journal of Clinical Investigation (2008) 118, 3, (1143-1153) DOI:10.1172/JCI33082) Journal of Clinical Investigation, 121 (3), pp. 1223. | Read more

Pfafferott K, Gaudieri S, Ulsenheimer A, James I, Heeg M, Nolan D, John M, Rauch A et al. 2011. Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity. PLoS One, 6 (2), pp. e16797. | Show Abstract | Read more

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

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Merani S, Petrovic D, James I, Chopra A, Cooper D, Freitas E, Rauch A, Di Iulio J et al. 2011. Effect of immune pressure on hepatitis C virus evolution: Insights from a single-source outbreak Hepatology, 53 (2), pp. 396-405. | Show Abstract | Read more

The host's immune response to hepatitis C virus (HCV) can result in the selection of characteristic mutations (adaptations) that enable the virus to escape this response. The ability of the virus to mutate at these sites is dependent on the incoming virus, the fitness cost incurred by the mutation, and the benefit to the virus in escaping the response. Studies examining viral adaptation in chronic HCV infection have shown that these characteristic immune escape mutations can be observed at the population level as human leukocyte antigen (HLA)-specific viral polymorphisms. We examined 63 individuals with chronic HCV infection who were infected from a single HCV genotype 1b source. Our aim was to determine the extent to which the host's immune pressure affects HCV diversity and the ways in which the sequence of the incoming virus, including preexisting escape mutations, can influence subsequent mutations in recipients and infection outcomes. Conclusion: HCV sequences from these individuals revealed 29 significant associations between specific HLA types within the new hosts and variations within their viruses, which likely represent new viral adaptations. These associations did not overlap with previously reported adaptations for genotypes 1a and 3a and possibly reflected a combination of constraint due to the incoming virus and genetic distance between the strains. However, these sites accounted for only a portion of the sites in which viral diversity was observed in the new hosts. Furthermore, preexisting viral adaptations in the incoming (source) virus likely influenced the outcomes in the new hosts. © 2011 American Association for the Study of Liver Diseases.

Hutchinson S, Sims S, O'Hara G, Silk J, Gileadi U, Cerundolo V, Klenerman P. 2011. A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus. PLoS One, 6 (2), pp. e14646. | Show Abstract | Read more

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8(+) T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8(+) T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8(+) T cell responses to MCMV - both conventional memory responses and those undergoing long-term expansion or "inflation". We infected LMP7(-/-) and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8(+) T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory "inflating" epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8(+) T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.

Thomson EC, Fleming VM, Main J, Klenerman P, Weber J, Eliahoo J, Smith J, McClure MO, Karayiannis P. 2011. Predicting spontaneous clearance of acute hepatitis C virus in a large cohort of HIV-1-infected men. Gut, 60 (6), pp. 837-845. | Show Abstract | Read more

OBJECTIVE: An epidemic of acute hepatitis C virus (HCV) infection in HIV-positive men-who-have-sex-with-men (MSM) is emerging in Europe, Australia and the USA. The aim of this study was to characterise the natural history of primary HCV in this setting and to assess host and viral factors which predict spontaneous clearance. METHODS: This prospective longitudinal cohort study was carried out in 112 HIV-positive patients who were followed in a single centre (the St Mary's Acute HCV Cohort). Plasma and peripheral blood mononuclear cells (PBMCs) were obtained at monthly intervals for 3 months and at 3-monthly intervals thereafter for a median of 45 months (IQR = 29-69 months). The primary end point was spontaneous clearance of HCV. Cox regression was used to assess the impact of clinical and virological variables on outcome, including liver function, CD4 count, rate of HCV RNA decline, T cell response and clonal sequence evolution within the HCV E2 envelope gene. RESULTS: 15% of patients cleared HCV spontaneously, while 85% progressed towards chronicity. The latter group included a significant proportion of 'fluctuating' progressors (37.5%), in whom a fall followed by a rise (>1 log₁₀) in viraemia was observed. This was associated with superinfection with new HCV strains and partially effective T cell responses. Spontaneous clearance was strongly associated with a 2.2 log₁₀ viral load drop within 100 days of infection (HR = 1.78; p < 0.0001), elevated bilirubin (≥ 40 μmol/l; HR = 5.04; p = 0.006), elevated alanine aminotransferase (ALT; ≥ 1000 IU/ml; HR = 2.62; p = 0.048) and baseline CD4 count ≥ 650 × 10⁶/l (HR = 2.66; p = 0.045), and only occurred in patients with genotype 1 infection. Evolution to spontaneous clearance occurred in patients with low viral diversity in the presence of an early multispecific T cell response. CONCLUSIONS: Spontaneous clearance of acute HCV in HIV-positive men can be predicted by a rapid decline in viral load, high CD4 count, elevated bilirubin and ALT, and is associated with low viral diversity and strong T cell responses.

Merani S, Petrovic D, James I, Chopra A, Cooper D, Freitas E, Rauch A, di Iulio J et al. 2011. Effect of immune pressure on hepatitis C virus evolution: insights from a single-source outbreak. Hepatology, 53 (2), pp. 396-405. | Show Abstract | Read more

UNLABELLED: The host's immune response to hepatitis C virus (HCV) can result in the selection of characteristic mutations (adaptations) that enable the virus to escape this response. The ability of the virus to mutate at these sites is dependent on the incoming virus, the fitness cost incurred by the mutation, and the benefit to the virus in escaping the response. Studies examining viral adaptation in chronic HCV infection have shown that these characteristic immune escape mutations can be observed at the population level as human leukocyte antigen (HLA)-specific viral polymorphisms. We examined 63 individuals with chronic HCV infection who were infected from a single HCV genotype 1b source. Our aim was to determine the extent to which the host's immune pressure affects HCV diversity and the ways in which the sequence of the incoming virus, including preexisting escape mutations, can influence subsequent mutations in recipients and infection outcomes. CONCLUSION: HCV sequences from these individuals revealed 29 significant associations between specific HLA types within the new hosts and variations within their viruses, which likely represent new viral adaptations. These associations did not overlap with previously reported adaptations for genotypes 1a and 3a and possibly reflected a combination of constraint due to the incoming virus and genetic distance between the strains. However, these sites accounted for only a portion of the sites in which viral diversity was observed in the new hosts. Furthermore, preexisting viral adaptations in the incoming (source) virus likely influenced the outcomes in the new hosts.

Smith JA, Aberle JH, Fleming VM, Ferenci P, Thomson EC, Karayiannis P, McLean AR, Holzmann H, Klenerman P. 2010. Dynamic coinfection with multiple viral subtypes in acute hepatitis C. J Infect Dis, 202 (12), pp. 1770-1779. | Show Abstract | Read more

INTRODUCTION: Acute hepatitis C virus (HCV) infection is rarely studied, but virus sequence evolution and host-virus dynamics during this early stage may influence the outcome of infection. Hypervariable region 1 (HVR1) is genetically diverse and under selective pressure from the host immune response. We analyzed HVR1 evolution by frequent sampling of an acutely infected HCV cohort. METHODS: Three or more pretreatment samples were obtained from each of 10 acutely infected subjects. Polymerase chain reaction amplification was performed with multiple primer combinations to identify the full range of sequences present. Positive samples were cloned and sequenced. Phylogenetic analyses were used to assess viral diversity. RESULTS: Eight of the 10 subjects were coinfected with at least 2 HCV subtypes. Multiple subtypes were detected in individual samples, and their relative proportions changed through acute infection. The subjects with the most complex subtype structure also had a dynamic viral load; however, changes in viral load were not directly linked to changes in subtype. CONCLUSIONS: This well-sampled cohort with acute HCV infection was characterized by dynamic coinfection with multiple viral subtypes, representing a highly complex virologic landscape extremely early in infection.

Huang KH, Bonsall D, Katzourakis A, Thomson EC, Fidler SJ, Main J, Muir D, Weber JN et al. 2010. B-cell depletion reveals a role for antibodies in the control of chronic HIV-1 infection. Nat Commun, 1 (7), pp. 102. | Show Abstract | Read more

HIV can be partially contained by host immunity and understanding the basis of this may inform vaccine design. The importance of B-cell function in long-term control is poorly understood. One method of investigating this is in vivo cellular depletion. In this study, we take advantage of a unique opportunity to investigate the role of B cells in an HIV-infected patient. The HIV-1(+) patient studied here was not taking antiretroviral drugs and was treated for pre-existing low-grade lymphoplasmacytoid lymphoma by depletion of CD20+ B cells using rituximab. We demonstrate that B-cell depletion results in a decline in autologous neutralizing antibody (NAb) responses and a 1.7 log(10) rise in HIV-1 plasma viral load (pVL). The recovery of NAbs results in a decline in pVL. The HIV-1 sequences diversify and NAb-resistant mutants are subsequently selected. These data suggest that B-cell function can contribute to the long-term control of pVL, and that NAbs may be more important in controlling chronic HIV-1 infection than previously suspected.

Woodhouse SD, Narayan R, Latham S, Lee S, Antrobus R, Gangadharan B, Luo S, Schroth GP, Klenerman P, Zitzmann N. 2010. Transcriptome sequencing, microarray, and proteomic analyses reveal cellular and metabolic impact of hepatitis C virus infection in vitro. Hepatology, 52 (2), pp. 443-453. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels. CONCLUSION: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus-host interaction which may be highly relevant to pathogenesis in vivo.

Rohrbach J, Robinson N, Harcourt G, Hammond E, Gaudieri S, Gorgievski M, Telenti A, Keiser O et al. 2010. Cellular immune responses to HCV core increase and HCV RNA levels decrease during successful antiretroviral therapy. Gut, 59 (9), pp. 1252-1258. | Show Abstract | Read more

BACKGROUND: Hepatitis C virus (HCV) infection is a major cause of morbidity in HIV infected individuals. Coinfection with HIV is associated with diminished HCV-specific immune responses and higher HCV RNA levels. AIMS: To investigate whether long-term combination antiretroviral therapy (cART) restores HCV-specific T cell responses and improves the control of HCV replication. METHODS: T cell responses were evaluated longitudinally in 80 HIV/HCV coinfected individuals by ex vivo interferon-gamma-ELISpot responses to HCV core peptides, that predominantly stimulate CD4(+) T cells. HCV RNA levels were assessed by real-time PCR in 114 individuals. RESULTS: The proportion of individuals with detectable T cell responses to HCV core peptides was 19% before starting cART, 24% in the first year on cART and increased significantly to 45% and 49% after 33 and 70 months on cART (p=0.001). HCV-specific immune responses increased in individuals with chronic (+31%) and spontaneously cleared HCV infection (+30%). Median HCV RNA levels before starting cART were 6.5 log(10) IU/ml. During long-term cART, median HCV-RNA levels slightly decreased compared to pre-cART levels (-0.3 log10 IU/ml, p=0.02). CONCLUSIONS: Successful cART is associated with increasing cellular immune responses to HCV core peptides and with a slight long-term decrease in HCV RNA levels. These findings are in line with the favourable clinical effects of cART on the natural history of hepatitis C and with the current recommendation to start cART earlier in HCV/HIV coinfected individuals.

Sims S, Willberg C, Klenerman P. 2010. MHC-peptide tetramers for the analysis of antigen-specific T cells. Expert Rev Vaccines, 9 (7), pp. 765-774. | Show Abstract | Read more

The development of the fluorescently labeled tetrameric MHC-peptide complex has enabled the direct visualization, quantification and phenotypic characterization of antigen-specific T cells using flow cytometry and has transformed our understanding of cellular immune responses. The combination of this technology with functional assays provides many new insights into these cells, allowing investigation into their lifecycle, manner of death and effector function. In this article, we hope to provide an overview of the techniques used in the construction of these tetramers, the problems and solutions associated with them, and the methods used in the study of antigen-specific T cells. Understanding how the antigen-specific cells develop and function in different circumstances and with different pathogens will be key to understanding natural host defense, as well as vaccine design and assessment.

Komatsu H, Inui A, Sogo T, Hiejima E, Tateno A, Klenerman P, Fujisawa T. 2010. Cellular immunity in children with successful immunoprophylactic treatment for mother-to-child transmission of hepatitis B virus. BMC Infect Dis, 10 (1), pp. 103. | Show Abstract | Read more

BACKGROUND: The administration of hepatitis B immunoglobulin followed by hepatitis B vaccine can result in a protective efficacy of almost 90% in mother-to-child transmission of hepatitis B virus (HBV). However, little is known about immunity against HBV infection in children after immunoprophylactic treatment. We tried to assess the association between T-cell responses and viremia in children after successful prophylactic treatment. METHODS: Thirteen children and their 8 HBV carrier mothers (8 families), who were positive for human leukocyte antigen (HLA)-A24, were enrolled in this study. All of the 13 children received immunoprophylactic treatment and became negative for hepatitis B surface antigen (HBsAg) after birth. HBV-specific cytotoxic T lymphocyte (CTL) responses were evaluated using IFNgamma - enzyme-linked immunosorbent spot (ELISPOT) and major histocompatibility complex class I peptide pentamer assays. Serum HBV DNA was measured by real-time PCR. RESULTS: Significant HBV-specific T-cell responses were detected in 2 (15%) of the 13 children by ELISPOT. However, the frequency of HLA-A24-HBV-specific CTLs was very low in both HBV carrier mothers and children using pentamers. Of the 13 children, 4 (31%) were positive for serum HBV DNA. However, the levels of serum HBV DNA were 100 copies/ml or less. One of the 2 children in whom significant HBV-specific CTL responses were detectable was positive for serum HBV DNA. CONCLUSIONS: HBV core and polymerase-specific T-cell responses were detected and a low-dose viremia was observed in children after successful immunoprophylaxis treatment. Although the presence of viremia was not related to HBV-specific T-cell responses, CTLs might play a role in the control of HBV infection in children born to HBsAg-positive mothers after immunoprophylactic treatment.

Syhavong B, Rasachack B, Smythe L, Rolain JM, Roque-Afonso AM, Jenjaroen K, Soukkhaserm V, Phongmany S et al. 2010. The infective causes of hepatitis and jaundice amongst hospitalised patients in Vientiane, Laos. Trans R Soc Trop Med Hyg, 104 (7), pp. 475-483. | Show Abstract | Read more

There is little information on the diverse infectious causes of jaundice and hepatitis in the Asiatic tropics. Serology (hepatitis A, B, C and E, leptospirosis, dengue, rickettsia), antigen tests (dengue), PCR assays (hepatitis A, C and E) and blood cultures (septicaemia) were performed on samples from 392 patients admitted with jaundice or raised transaminases (> or =x3) to Mahosot Hospital, Vientiane, Laos over 3 years. Conservative definitions suggested diagnoses of dengue (8.4%), rickettsioses (7.3%), leptospirosis (6.8%), hepatitis B (4.9%), hepatitis C (4.9%), community-acquired septicaemia (3.3%) and hepatitis E (1.6%). Although anti-hepatitis A virus (HAV) IgM antibody results suggested that 35.8% of patients had acute HAV infections, anti-HAV IgG antibody avidity and HAV PCR suggested that 82% had polyclonal activation and not acute HAV infections. Scrub typhus, murine typhus or leptospirosis were present in 12.8% of patients and were associated with meningism and relatively low AST and ALT elevation. These patients would be expected to respond to empirical doxycycline therapy which, in the absence of virological diagnosis and treatment, may be an appropriate cost-effective intervention in Lao patients with jaundice/hepatitis.

Mueller M, Spangenberg HC, Kersting N, Altay T, Blum HE, Klenerman P, Thimme R, Semmo N. 2010. Virus-specific CD4+ T cell responses in chronic HCV infection in blood and liver identified by antigen-specific upregulation of CD154. J Hepatol, 52 (6), pp. 800-811. | Show Abstract | Read more

BACKGROUND & AIMS: Virus-specific CD4+ T cells play a major role in hepatitis C virus (HCV) infection. Viral clearance is associated with vigorous and multispecific CD4+ T cell responses, while chronic infection has been shown to be associated with weak or absent T cell responses. Most of these studies, however, have used functional assays to analyse virus-specific CD4+ T cell responses. Therefore, the important question, of whether virus-specific CD4+ T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analysed in detail. METHODS: To address this issue, a novel assay, where CD4+ T cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to HCV antigens, was used in a cohort of chronically infected HCV patients and patients who spontaneously resolved HCV infection. These responses were compared to functional assays, such as the IFN-gamma ELISpot and flow cytometry-based proliferative assays. RESULTS: Our results reveal that using the CD154 assay, virus-specific CD4+ T cells are readily detectable during chronic HCV infection albeit at a lower frequency when compared to patients who spontaneously resolved the infection. Importantly, no CD4+ T cell responses were detectable from these patients when using functional assays. Finally, these cell populations were enriched in the intrahepatic compartment. CONCLUSIONS: Our findings suggest that HCV-specific CD4+ T cell responses are readily detectable in chronic HCV infection and enriched in the infected liver.

Thomas AC, Forster MR, Bickerstaff AA, Zimmerman PD, Wing BA, Trgovcich J, Bergdall VK, Klenerman P, Cook CH. 2010. Occult cytomegalovirus in vivarium-housed mice may influence transplant allograft acceptance. Transpl Immunol, 23 (1-2), pp. 86-91. | Show Abstract | Read more

We have recently shown that latent murine cytomegalovirus (MCMV) can influence murine transplant allograft acceptance. During these studies we became aware that vivarium-housed control mice can acquire occult MCMV infection. The purpose of this investigation was to confirm occult MCMV transmission and determine the timing, vehicle, and possible consequences of transmission. Mice arriving from a commercial vendor were negative for MCMV both by commercial serologic testing and by our nested PCR. Mice housed in our vivarium became positive for MCMV DNA 30-60 days after arrival, but remained negative for MCMV by commercial serologic testing. To confirm MCMV we sequenced PCR products for several genes and showed >99% homology to MCMV. Further sequence analyses show that the occult MCMV is similar to a laboratory strain of MCMV, but the vehicle of transmission remains unclear. Control tissues from historical experiments with unexplained graft losses were evaluated for occult MCMV, and mice with unexplained allograft losses showed significantly higher incidence of occult MCMV than did allograft acceptors. Deliberate infection with very low titer MCMV confirmed that viral transmission can occur without measurable virus specific antibody or T-cell responses. These data suggest that vivarium-housed mice can develop occult MCMV that is missed by currently available commercial serologic testing, and that these infections may influence transplant allograft acceptance.

Humphreys I, Fleming V, Fabris P, Parker J, Schulenberg B, Brown A, Demetriou C, Gaudieri S et al. 2010. Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection (vol 83, pg 11456, 2009) JOURNAL OF VIROLOGY, 84 (3), pp. 1664-1664. | Read more

Billerbeck E, Kang YH, Walker L, Lockstone H, Grafmueller S, Fleming V, Flint J, Willberg CB et al. 2010. Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties. Proc Natl Acad Sci U S A, 107 (7), pp. 3006-3011. | Show Abstract | Read more

CD8(+) T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor gamma-t, P = 6 x 10(-9); RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 x 10(-7); IL-18 receptor, P = 4 x 10(-6)), and chemokine receptors (e.g., CCR6, P = 3 x 10(-8); CXCR6, P = 3 x 10(-7); CCR2, P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

Fleming VM, Harcourt G, Barnes E, Klenerman P. 2010. Virological footprint of CD4+ T-cell responses during chronic hepatitis C virus infection. J Gen Virol, 91 (Pt 6), pp. 1396-1406. | Show Abstract | Read more

Human and animal model evidence suggests that CD4+ T cells play a critical role in the control of chronic hepatitis C virus (HCV) infection. However, despite their importance, the mechanism behind the failure of such populations in chronic disease is not understood and the contribution of viral mutation is not known. To address this, this study defined the specificity and virological footprint of CD4+ T cells in chronic infection. CD8+ T-cell-depleted peripheral blood mononuclear cells from 61 HCV genotype 1-infected patients were analysed against a panel of peptides covering the HCV genotype 1 core--a region where CD4+ T-cell responses may be reproducibly obtained. In parallel, the core region and E2 protein were sequenced. Gamma interferon-secreting CD4+ T-cell responses directed against multiple epitopes were detected in 53% of individuals, targeting between one and four peptides in the HCV core. Viral sequence evaluation revealed that these CD4+ T-cell responses were associated with mutants in 2/21 individuals. In these two cases, the circulating sequence variant was poorly recognized by host CD4+ T cells. Bioinformatics analyses revealed no overall evidence of selection in the target epitopes and no differences between the groups with and without detectable CD4+ T-cell responses. It was concluded that sustained core peptide-specific CD4+ T-cell responses may be reproducibly measured during chronic HCV infection and that immune escape may occur in specific instances. However, overall the virological impact of such responses is limited and other causes for CD4+ T-cell failure in HCV must be sought.

Prendergast A, Prado JG, Kang YH, Chen F, Riddell LA, Luzzi G, Goulder P, Klenerman P. 2010. HIV-1 infection is characterized by profound depletion of CD161+ Th17 cells and gradual decline in regulatory T cells. AIDS, 24 (4), pp. 491-502. | Show Abstract | Read more

OBJECTIVE: CD4 T-cell depletion is central to HIV pathogenesis. However, the relative impact of HIV on Th17 and regulatory T cell (Treg) subsets remains unclear. CD161 CD4 cells are a recently identified, gut-homing Th17 precursor population. The balance between pro-inflammatory Th17 and immunoregulatory Tregs may be critical in HIV pathogenesis. This study addressed changes in CD161, Th17 and Treg subsets during untreated HIV infection. METHODS: Peripheral blood mononuclear cells were isolated from HIV-infected and HIV-uninfected individuals and stained to characterize CD161 CD4 cells, Th17 cells [by elaboration of interleukin (IL)-17A], Tregs (CD3CD4CD25FoxP3 cells) and CD8 activation (CD38/HLA-DR cells). In-vitro infectability of CD161 and Th17 cells by HIV was assessed in healthy donor CD4 cells by intracellular p24 expression. RESULTS: Peripheral blood Th17 cells were depleted 10-fold in HIV-infected, compared to HIV-uninfected individuals (P < 0.0001) across a range of disease stages, accompanied by a significant reduction of CD161 T cells (P = 0.024). Both Th17 cells and CD161 CD4 T cells were permissive to HIV replication in vitro. Profound loss of Th17 cells before the onset of advanced disease contrasted with a gradual decline in absolute Tregs during HIV disease progression in untreated individuals followed longitudinally (R = 0.71, P = 0.003). Loss of Tregs was associated with increased immune activation (R = -0.33, P = 0.03). CONCLUSION: HIV-infected individuals showed profound loss of Th17 cells, which may impair mucosal immunity, and reduced CD161 CD4 cells, which may limit Th17 reconstitution. A gradual decline in Tregs during disease progression was associated with increased immune activation.

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Huang KHG, Bonsall D, Katzourakis A, Thomson EC, Fidler SJ, Main J, Muir D, Weber JN et al. 2010. B-cell depletion reveals a role for antibodies in the control of chronic HIV-1 infection Nature Communications, 1 (7), | Show Abstract | Read more

HIV can be partially contained by host immunity and understanding the basis of this may inform vaccine design. The importance of B-cell function in long-term control is poorly understood. One method of investigating this is in vivo cellular depletion. In this study, we take advantage of a unique opportunity to investigate the role of B cells in an HIV-infected patient. The HIV-1+ patient studied here was not taking antiretroviral drugs and was treated for pre-existing low-grade lymphoplasmacytoid lymphoma by depletion of CD20+ B cells using rituximab. We demonstrate that B-cell depletion results in a decline in autologous neutralizing antibody (NAb) responses and a 1.7 log10 rise in HIV-1 plasma viral load (pVL). The recovery of NAbs results in a decline in pVL. The HIV-1 sequences diversify and NAb-resistant mutants are subsequently selected. These data suggest that B-cell function can contribute to the long-term control of pVL, and that NAbs may be more important in controlling chronic HIV-1 infection than previously suspected. © 2010 Macmillan Publishers Limited. All rights reserved.

Huang GJ, Smith AL, Gray DH, Cosgrove C, Singer BH, Edwards A, Sims S, Parent JM et al. 2010. A genetic and functional relationship between T cells and cellular proliferation in the adult hippocampus. PLoS Biol, 8 (12), pp. e1000561. | Show Abstract | Read more

Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.

Humphreys I, Fleming V, Fabris P, Parker J, Schulenberg B, Brown A, Demetriou C, Gaudieri S et al. 2010. Full-length characterization of hepatitis C virus subtype 3a reveals novel hypervariable regions under positive selection during acute infection (Journal of Virology (2009) 83, 22 (11456-11466)) Journal of Virology, 84 (3), pp. 1664. | Read more

Klapa S, Mueller A, Csernok E, Fagin U, Klenerman P, Holl-Ulrich K, Gross WL, Lamprecht P. 2010. Lower numbers of FoxP3 and CCR4 co-expressing cells in an elevated subpopulation of CD4(+)CD25(high) regulatory T cells from Wegener's granulomatosis CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, 28 (1), pp. S72-S80.

Klapa S, Mueller A, Csernok E, Fagin U, Klenerman P, Holl-Ulrich K, Gross WL, Lamprecht P. 2010. Lower numbers of FoxP3 and CCR4 co-expressing cells in an elevated subpopulation of CD4+CD25high regulatory T cells from Wegener's granulomatosis. Clin Exp Rheumatol, 28 (1 Suppl 57), pp. 72-80. | Show Abstract

Defects in regulatory T (Treg) cells have been implicated in the pathogenesis of chronic inflammatory and autoimmune diseases, such as Wegener's granulomatosis (WG). This study aimed at evaluating numbers, phenotype and suppressive capacity of Treg cells in WG. Peripheral blood (PB) mononuclear cells from 22 WG-patients (17 active, 5 remission) and 22 sex- and age-matched healthy controls (HC) were examined for Treg cells by flow cytometry measuring CD4, CD25, transcription factor forkhead box P3 (FoxP3), chemokine receptor CCR4 and interferon receptor I (IFNRI). Suppressive function of CD4+CD25high Treg cells from 3 WG-patients and 3 HC was analysed using a carboxyfluoresceindiacetate-succinimidylester-based in vitro proliferation assay. Endonasal biopsies of 10 WG- and 5 sinusitis-patients were investigated for CD3+FoxP3+ cells, employing double immunohistochemistry. WG-patients displayed elevated numbers of CD4+CD25med T cells and of CD4+CD25high Treg cells. CD4+ T cells of WG-patients contained higher numbers of CCR4+ cells. However, CD4+CD25high Treg cells of WG-patients exhibited decreased numbers of cells co-expressing FoxP3 and CCR4. A low but significant increase of CD4+CD25highIFNRI+ Treg cells was detected in WG-patients. 9 days following stimulation with interferon (IFN)alpha + proteinase 3 (PR3), a reduced suppression of proliferation of responder T cells was observed for WG and proliferated CD4+CD25high Treg cells still showed downregulated co-expressions of FoxP3 and CCR4. Wegener's granuloma exhibited increased numbers of CD3+FoxP3+ cells. The results indicate upregulated numbers of Treg cells in PB and nasal mucosa as well as phenotypical and functional alterations of PB Treg cells in WG, some presumably mediated through PR3 and IFN-alpha.

Yalcin B, Nicod J, Bhomra A, Davidson S, Cleak J, Farinelli L, Østerås M, Whitley A et al. 2010. Commercially available outbred mice for genome-wide association studies. PLoS Genet, 6 (9), pp. e1001085. | Show Abstract | Read more

Genome-wide association studies using commercially available outbred mice can detect genes involved in phenotypes of biomedical interest. Useful populations need high-frequency alleles to ensure high power to detect quantitative trait loci (QTLs), low linkage disequilibrium between markers to obtain accurate mapping resolution, and an absence of population structure to prevent false positive associations. We surveyed 66 colonies for inbreeding, genetic diversity, and linkage disequilibrium, and we demonstrate that some have haplotype blocks of less than 100 Kb, enabling gene-level mapping resolution. The same alleles contribute to variation in different colonies, so that when mapping progress stalls in one, another can be used in its stead. Colonies are genetically diverse: 45% of the total genetic variation is attributable to differences between colonies. However, quantitative differences in allele frequencies, rather than the existence of private alleles, are responsible for these population differences. The colonies derive from a limited pool of ancestral haplotypes resembling those found in inbred strains: over 95% of sequence variants segregating in outbred populations are found in inbred strains. Consequently it is possible to impute the sequence of any mouse from a dense SNP map combined with inbred strain sequence data, which opens up the possibility of cataloguing and testing all variants for association, a situation that has so far eluded studies in completely outbred populations. We demonstrate the colonies' potential by identifying a deletion in the promoter of H2-Ea as the molecular change that strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes.

Forster MR, Trgovcich J, Zimmerman P, Chang A, Miller C, Klenerman P, Cook CH. 2010. Antiviral prevention of sepsis induced cytomegalovirus reactivation in immunocompetent mice. Antiviral Res, 85 (3), pp. 496-503. | Show Abstract | Read more

INTRODUCTION: Immunocompetent patients can reactivate latent cytomegalovirus (CMV) during critical illness and reactivation is associated with significantly worse outcomes. Prior to clinical trials in humans to prove causality, we sought to determine an optimal antiviral treatment strategy. METHODS: Mice latently infected with murine CMV (MCMV) received a septic reactivation trigger and were randomized to receive one of four ganciclovir regimens or saline. Lungs were evaluated for viral transcriptional reactivation and fibrosis after each regimen. Influences of ganciclovir on early sepsis-induced pulmonary inflammation and T-cell activation were studied after sepsis induction. RESULTS: All ganciclovir regimens reduced measurable MCMV transcriptional reactivation, and 10mg/day for 7 or 21 days was most effective. Lower dose (5mg/kg/day) or delayed therapy was associated with significant breakthrough reactivation. Higher doses of ganciclovir given early were associated with the lowest incidence of pulmonary fibrosis, and delay of therapy for 1 week was associated with significantly worse pulmonary fibrosis. Although bacterial sepsis induced activation of MCMV-specific pulmonary T-cells, this activation was not influenced by ganciclovir. CONCLUSION: These results suggest that antiviral treatment trials in humans should use 10mg/kg/day ganciclovir administered as early as possible in at-risk patients to minimize reactivation events and associated pulmonary injury.

Jones L, Malavige G, Jeffery K, Kemp E, Breuer J, Klenerman P, Ogg GS. 2009. Tracking epitope-specific antiviral CD4+ T cell responses to a live attenuated vaccine reveals ongoing functional responses. Vaccine, 27 (52), pp. 7398-7401. | Show Abstract | Read more

There are few studies that have examined the frequencies of epitope-specific CD4(+) T cells following the use of a highly effective vaccine, yet such data would potentially be of value for the development of novel vaccination strategies. In this study we tracked human epitope-specific CD4(+) T cell responses over time after immunisation with a live attenuated varicella zoster virus vaccine by MHC Class II tetrameric complexes and functional assays. We show that the peptide-specific responses reflect those against whole virus antigens, and are similar in both frequency and phenotype to those found in healthy volunteers, despite a highly attenuated and clinically inapparent infection.

Walker LJ, Sewell AK, Klenerman P. 2010. T cell sensitivity and the outcome of viral infection. Clin Exp Immunol, 159 (3), pp. 245-255. | Show Abstract | Read more

The importance of CD8(+) T cells in the control of viral infections is well established. However, what differentiates CD8(+) T cell responses in individuals who control infection and those who do not is not well understood. 'Functional sensitivity' describes an important quality of the T cell response and is determined in part by the affinity of the T cell receptor for antigen. A more sensitive T cell response is generally believed to be more efficient and associated with better control of viral infection, yet may also drive viral mutation and immune escape. Various in vitro techniques have been used to measure T cell sensitivity; however, rapid ex vivo analysis of this has been made possible by the application of the 'magic' tetramer technology. Such tools have potentially important applications in the design and evaluation of vaccines.

Fitzmaurice K, Petrovic D, Gaudieri S, Freitas E, Sims S, Long A, Kelleher D, Klenerman P. 2009. HLA FOOTPRINTING REVEALS A NOVEL PROTECTIVE CD8+T CELL RESPONSE IN HEPATITIS C VIRUS INFECTION JOURNAL OF INFECTION, 59 (6), pp. S428-S429.

Kasprowicz V, Kang YH, Lucas M, Schulze zur Wiesch J, Kuntzen T, Fleming V, Nolan BE, Longworth S et al. 2010. Hepatitis C virus (HCV) sequence variation induces an HCV-specific T-cell phenotype analogous to spontaneous resolution. J Virol, 84 (3), pp. 1656-1663. | Show Abstract | Read more

Hepatitis C virus (HCV)-specific CD8(+) T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127(+) T cells is driven by viral variation.

Humphreys I, Fleming V, Fabris P, Parker J, Schulenberg B, Brown A, Demetriou C, Gaudieri S et al. 2009. Full-length characterization of hepatitis C virus subtype 3a reveals novel hypervariable regions under positive selection during acute infection. J Virol, 83 (22), pp. 11456-11466. | Show Abstract | Read more

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.

Dunn C, Peppa D, Khanna P, Nebbia G, Jones M, Brendish N, Lascar RM, Brown D et al. 2009. Temporal analysis of early immune responses in patients with acute hepatitis B virus infection. Gastroenterology, 137 (4), pp. 1289-1300. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis B virus (HBV) causes more than 1 million deaths annually from immune-mediated liver damage. The long incubation period has been difficult to study; by the time most patients present, massive viremia and the majority of viral clearance have already occurred. The aim of this study was to investigate the contribution of innate and adaptive immune mechanisms in early acute HBV through access to an unusual cohort of patients sampled in the preclinical phase and followed up to resolution of their infection. METHODS: Twenty-one patients with acute HBV were studied, 8 of them from before the peak of viremia. Circulating innate cytokines were quantitated by enzyme-linked immunosorbent assay and natural killer (NK) and T-cell effector function by flow cytometry. Results were correlated with temporal changes in viral load, serology, and liver inflammation and compared with healthy controls. RESULTS: Type I interferon (IFN) remained barely detectable throughout, with concentrations no higher than those found in healthy controls. Similarly, interleukin-15 and IFN-lambda1 were not induced during peak viremia. NK cell activation and capacity for IFN-gamma production were reduced at peak viremia. Early functional HBV-specific CD4 and CD8 T-cell responses were attenuated as viral load increased and recovered again as infection resolved. The transient inhibition of NK and T-cell responses coincided with a surge in the immunosuppressive cytokine interleukin-10 accompanying HBV viremia. CONCLUSIONS: The early stages of acute HBV are characterized by induction of interleukin-10 rather than type I IFN, accompanied by a temporary attenuation of NK and T-cell responses.

Barnes E, Antonella F, Aston S, Smith K, Brown AC, Capone S, Ambrosio M, Harrison GAL et al. 2009. PHASE I TRIAL OF A HIGHLY IMMUNOGENIC T-CELL VACCINE FOR HEPATITIS C VIRUS BASED ON NOVEL ADENOVIRAL VECTORS FROM RARE SEROTYPES HEPATOLOGY, 50 (4), pp. 397A-398A.

Rauch A, James I, Pfafferott K, Nolan D, Klenerman P, Cheng W, Mollison L, McCaughan G et al. 2009. Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure. Hepatology, 50 (4), pp. 1017-1029. | Show Abstract | Read more

UNLABELLED: Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

Sharp CP, Lail A, Donfield S, Simmons R, Leen C, Klenerman P, Delwart E, Gomperts ED, Simmonds P. 2009. High frequencies of exposure to the novel human parvovirus PARV4 in hemophiliacs and injection drug users, as detected by a serological assay for PARV4 antibodies. J Infect Dis, 200 (7), pp. 1119-1125. | Show Abstract | Read more

BACKGROUND: PARV4 is a human parvovirus that was first detected in and cloned from an individual with a human immunodeficiency virus (HIV) seroconversion-like illness and that subsequently persisted in the lymphoid tissue and bone marrow. In contrast to human parvovirus B19 infections, PARV4 infections are most frequently detected in injection drug users (IDUs), particularly those who are coinfected with HIV type 1 (HIV-1). To investigate the routes of transmission of PARV4 and to ascertain whether infections are acquired through plasma-derived blood products, we developed a novel anti-PARV4 enzyme-linked immunosorbent assay (ELISA) to determine its seroprevalence in subjects with parenteral exposure. METHODS: PARV4 viral protein 2 (VP2) was expressed and used as antigen in an indirect ELISA, to detect anti-PARV4 immunoglobulin G. RESULTS: All 50 adult control subjects who were nonparenterally exposed to PARV4 were anti-PARV4 negative, in contrast to HIV-infected and HIV-uninfected IDUs, who had antibody frequencies of 67% and 33%, respectively. Predominantly parenteral transmission was confirmed by the finding of similar frequencies of infection among HIV-coinfected and HIV-uninfected hemophiliacs (11 of 20 individuals and 4 of 15 individuals, respectively) who were treated with nonvirally inactivated factor VIII/factor IX, whereas all but 1 of the 35 nonhemophiliac siblings of these siblings were found to be seronegative (despite having close household contact). CONCLUSIONS: The present study provides convincing evidence that PARV4 is primarily transmitted parenterally. Evidence for widespread infection of hemophiliacs treated with nonvirally inactivated clotting factor creates fresh safety concerns for plasma-derived blood products, particularly because parvoviruses are relatively resistant to virus inactivation.

Todryk SM, Pathan AA, Keating S, Porter DW, Berthoud T, Thompson F, Klenerman P, Hill AV. 2009. The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming. Immunology, 128 (1), pp. 83-91. | Show Abstract | Read more

Maintenance of T-cell responses is an essential feature in protection from many infectious diseases that must be harnessed in vaccination. The relationship between effector T-cell responses and more durable and highly proliferative T-cell memory, particularly in humans, is not well understood. In this study, effector T-cell responses were measured by overnight ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), whereas memory T cells were measured by 10-day culture followed by IFN-gamma ELISPOT (cultured ELISPOT). We observed a significant correlation between IFN-gamma responses to CD4-stimulatory, but not to CD8-stimulatory, recall antigens measured by these assays, suggesting a divergence in regulation. In vaccine trial participants who received a prime-boost vaccination regimen comprising malaria antigens delivered by poxviruses, there was a correlation between ex vivo and cultured responses on day 7, but not 3 months post-vaccination, with the ratio of cultured : ex vivo response increasing over time. To compare responses revealed by cultured ELISPOT in more detail, tetramers comprising viral recall antigens were used to ascribe effector-memory and central-memory T-cell phenotypes through CCR7 and CD62L costaining. For CD8(+) responses the effector phenotype decreased during the initial culture period and memory populations remained high within the resulting 20-fold to 50-fold increased IFN-gamma-secreting or tetramer(+) population. This was less marked for CD4(+) responses, which had higher starting memory phenotype. Depletion of these central-memory T-cell populations generally ablated responses in cultured ELISPOT and reduced ex vivo responses. This study highlights differences between CD4(+) and CD8(+) effector and memory T cells, and the more complex phenotype of CD4(+) T cells.

Klenerman P, Fleming V, Barnes E. 2009. What are the prospects for controlling hepatitis C? PLoS Med, 6 (6), pp. e1000096. | Read more

Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, Chang CH, Harndahl M et al. 2009. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance. Nat Immunol, 10 (6), pp. 636-646. | Show Abstract | Read more

Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.

Matthews PC, Leslie AJ, Katzourakis A, Crawford H, Payne R, Prendergast A, Power K, Kelleher AD et al. 2009. HLA footprints on human immunodeficiency virus type 1 are associated with interclade polymorphisms and intraclade phylogenetic clustering. J Virol, 83 (9), pp. 4605-4615. | Show Abstract | Read more

The selection of escape mutations has a major impact on immune control of infections with viruses such as human immunodeficiency virus (HIV). Viral evasion of CD8(+) T-cell responses leaves predictable combinations of escape mutations, termed HLA "footprints." The most clearly defined footprints are those associated with HLA alleles that are linked with successful control of HIV, such as HLA-B*57. Here we investigated the extent to which HLA footprint sites in HIV type 1 (HIV-1) are associated with viral evolution among and within clades. First, we examined the extent to which amino acid differences between HIV-1 clades share identity with sites of HLA-mediated selection pressure and observed a strong association, in particular with respect to sites of HLA-B selection (P < 10(-6)). Similarly, the sites of amino acid variability within a clade were found to overlap with sites of HLA-selected mutation. Second, we studied the impact of HLA selection on interclade phylogeny. Removing the sites of amino acid variability did not significantly affect clade-specific clustering, reflecting the central role of founder effects in establishing distinct clades. However, HLA footprints may underpin founder strains, and we show that amino acid substitutions between clades alter phylogeny, underlining a potentially substantial role for HLA in driving ongoing viral evolution. Finally, we investigated the impact of HLA selection on within-clade phylogeny and demonstrate that even a single HLA allele footprint can result in significant phylogenetic clustering of sequences. In conclusion, these data highlight the fact that HLA can be a strong selection force for both intra- and interclade HIV evolution at a population level.

Kawashima Y, Pfafferott K, Frater J, Matthews P, Payne R, Addo M, Gatanaga H, Fujiwara M et al. 2009. Adaptation of HIV-1 to human leukocyte antigen class I. Nature, 458 (7238), pp. 641-645. | Show Abstract | Read more

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Fox J, Willberg C, Ziprin P, Goldin R, Weber J, McClure M, Klenerman P, Fidler S. 2009. The role of the gut mucosa in protection from HIV-1 in highly exposed persistently seronegative individuals (HEPS) HIV MEDICINE, 10 pp. 5-5.

Gaudieri S, Rauch A, Pfafferott K, Barnes E, Cheng W, McCaughan G, Shackel N, Jeffrey GP et al. 2009. Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy. Hepatology, 49 (4), pp. 1069-1082. | Show Abstract | Read more

UNLABELLED: The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P <or= 0.05) at known drug resistance sites. CONCLUSION: Drug and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host "immune resistance" in the STAT-C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C.

Barnes E, Gelderblom HC, Humphreys I, Semmo N, Reesink HW, Beld MG, van Lier RA, Klenerman P. 2009. Cellular immune responses during high-dose interferon-alpha induction therapy for hepatitis C virus infection. J Infect Dis, 199 (6), pp. 819-828. | Show Abstract | Read more

BACKGROUND: The effect that high-dose interferon (IFN)-alpha induction therapy for hepatitis C virus (HCV) infection has on cellular immune responses is currently unknown. METHODS: Thirty-one treatment-naive patients with chronic HCV infection received amantadine and ribavirin, combined with 6 weeks of high-dose IFN-alpha-2b induction therapy followed by weekly pegylated IFN-alpha-2b, for 24 or 48 weeks. Using IFN-gamma and interleukin (IL)-2 enzyme-linked immunospot (ELISpot) assays, we analyzed the pattern of cytokine secretion by structural and nonstructural HCV- and cytomegalovirus (CMV)-specific T cells before, during, and after therapy. RESULTS: HCV-specific T cell responses, which were predominantly IFN-gamma secreting and which correlated with alanine transaminase levels (r2 = 0.45; P = .001), were found before treatment in 10 of 15 patients with a sustained virological response (SVR) and in 11 of 16 in the non-SVR group. There was a striking loss of IFN-gamma and IL-2 HCV-specific T cells during therapy, predominantly in the SVR group. This response recovered after cessation of therapy, regardless of outcome. Suppression of CMV-specific T cell responses, in addition to total lymphocyte counts, was also observed. CONCLUSIONS: High-dose IFN-alpha induction therapy leads to a profound decline in IL-2- and IFN-gamma-secreting HCV- and CMV-specific T cells. These data indicate that restoration of T cell responses is unlikely to be causally linked to an early response or SVR to therapy.

Prendergast A, Prado JG, Kang Y, Chen F, Riddell LA, Luzzi G, Goulder P, Klenerman P. 2009. HIV-1 infection is characterized by early loss of CD161+Th17 cells and gradual decline in regulatory T cells RETROVIROLOGY, 6 (SUPPL. 3),

Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, Chang C, Harndahl M et al. 2009. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance RETROVIROLOGY, 6 (Suppl 3), pp. P252-P252. | Read more

Hoffmann M, Hickling S, Filby A, Wilburg C, Cole D, Turner A, Sims S, McLean A et al. 2009. Novel tetramer technology for the detection of high affinity CD8 T cells RETROVIROLOGY, 6 (Suppl 3), pp. P409-P409. | Read more

Barnes E, Gelderblom HC, Humphreys I, Semmo N, Reesink HW, Beld MG, van Lier RA, Klenerman P. 2009. Cellular Immune Responses during High-Dose Interferon-alpha Induction Therapy for Hepatitis C Virus Infection. J Infect Dis, 199 (6), pp. 819-828. | Show Abstract | Read more

Background. The effect that high-dose interferon (IFN)-alpha induction therapy for hepatitis C virus (HCV) infection has on cellular immune responses is currently unknown. Methods. Thirty-one treatment-naive patients with chronic HCV infection received amantadine and ribavirin, combined with 6 weeks of high-dose IFN-alpha-2b induction therapy followed by weekly pegylated IFN-alpha-2b, for 24 or 48 weeks. Using IFN-gamma and interleukin (IL)-2 enzyme-linked immunospot (ELISpot) assays, we analyzed the pattern of cytokine secretion by structural and nonstructural HCV- and cytomegalovirus (CMV)-specific T cells before, during, and after therapy. Results. HCV-specific T cell responses, which were predominantly IFN-gamma secreting and which correlated with alanine transaminase levels ([Formula: see text]; [Formula: see text]), were found before treatment in 10 of 15 patients with a sustained virological response (SVR) and in 11 of 16 in the non-SVR group. There was a striking loss of IFN-gamma and IL-2 HCV-specific T cells during therapy, predominantly in the SVR group. This response recovered after cessation of therapy, regardless of outcome. Suppression of CMV-specific T cell responses, in addition to total lymphocyte counts, was also observed. Conclusions. High-dose IFN-alpha induction therapy leads to a profound decline in IL-2- and IFN-gamma-secreting HCV- and CMV-specific T cells. These data indicate that restoration of T cell responses is unlikely to be causally linked to an early response or SVR to therapy.

Dazert E, Neumann-Haefelin C, Bressanelli S, Fitzmaurice K, Kort J, Timm J, McKiernan S, Kelleher D et al. 2009. Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27-restricted human immune response. J Clin Invest, 119 (2), pp. 376-386. | Show Abstract | Read more

There is an association between expression of the MHC class I molecule HLA-B27 and protection following human infection with either HIV or HCV. In both cases, protection has been linked to HLA-B27 presentation of a single immunodominant viral peptide epitope to CD8+ T cells. If HIV mutates the HLA-B27-binding anchor of this epitope to escape the protective immune response, the result is a less-fit virus that requires additional compensatory clustered mutations. Here, we sought to determine whether the immunodominant HLA-B27-restricted HCV epitope was similarly constrained by analyzing the replication competence and immunogenicity of different escape mutants. Interestingly, in most HLA-B27-positive patients chronically infected with HCV, the escape mutations spared the HLA-B27-binding anchor. Instead, the escape mutations were clustered at other sites within the epitope and had only a modest impact on replication competence. Further analysis revealed that the cluster of mutations is required for efficient escape because a combination of mutations is needed to impair T cell recognition of the epitope. Artificially introduced mutations at the HLA-B27-binding anchors were found to be either completely cross-reactive or to lead to substantial loss of fitness. These results suggest that protection by HLA-B27 in HCV infection can be explained by the requirement to accumulate a cluster of mutations within the immunodominant epitope to escape T cell recognition.

Webster DP, Klenerman P, Collier J, Jeffery KJ. 2009. Development of novel treatments for hepatitis C. Lancet Infect Dis, 9 (2), pp. 108-117. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is a major and growing global health problem, affecting about 170 million people worldwide, and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Currently, treatment is restricted to interferon alfa and ribavirin, which leads to a successful outcome in only about 50% of individuals. New effective treatments with tolerable side-effect profiles are needed urgently, but development has been hindered by an inability to culture HCV and a scarcity of animal models. Herein, we review progress in HCV biology, including cell culture and new animal models, and the contribution of this work to our understanding of the virus' life-cycle and pathogenesis and development of specifically targeted antiviral treatment. We also discuss changes in our understanding of HCV epidemiology, clinical manifestations, and diagnostics.

Pybus OG, Barnes E, Taggart R, Lemey P, Markov PV, Rasachak B, Syhavong B, Phetsouvanah R et al. 2009. Genetic history of hepatitis C virus in East Asia. J Virol, 83 (2), pp. 1071-1082. | Show Abstract | Read more

The hepatitis C virus (HCV), which currently infects an estimated 3% of people worldwide, has been present in some human populations for several centuries, notably HCV genotypes 1 and 2 in West Africa and genotype 6 in Southeast Asia. Here we use newly developed methods of sequence analysis to conduct the first comprehensive investigation of the epidemic and evolutionary history of HCV in Asia. Our analysis includes new HCV core (n = 16) and NS5B (n = 14) gene sequences, obtained from serum samples of jaundiced patients from Laos. These exceptionally diverse isolates were analyzed in conjunction with all available reference strains using phylogenetic and Bayesian coalescent methods. We performed statistical tests of phylogeographic structure and applied a recently developed "relaxed molecular clock" approach to HCV for the first time, which indicated an unexpectedly high degree of rate variation. Our results reveal a >1,000-year-long development of genotype 6 in Asia, characterized by substantial phylogeographic structure and two distinct phases of epidemic history, before and during the 20th century. We conclude that HCV lineages representing preexisting and spatially restricted strains were involved in multiple, independent local epidemics during the 20th century. Our analysis explains the generation and maintenance of HCV diversity in Asia and could provide a template for further investigations of HCV spread in other regions.

Huang KH, Goedhals D, Fryer H, van Vuuren C, Katzourakis A, De Oliveira T, Brown H, Cassol S et al. 2009. Prevalence of HIV type-1 drug-associated mutations in pre-therapy patients in the Free State, South Africa. Antivir Ther, 14 (7), pp. 975-984. | Show Abstract | Read more

BACKGROUND: We aimed to characterize the molecular epidemiology of HIV type-1 (HIV-1) and the prevalence of drug-associated mutations prior to initiating highly active antiretroviral therapy (HAART) in the Free State province, South Africa. The Free State has a population of 3 million, an antenatal HIV prevalence of approximately 34% and a well established infrastucture for antiretroviral (ARV) provision. METHODS: HIV-1 polymerase genes were sequenced from 425 HAART-naive HIV-1-positive patients at voluntary primary healthcare HIV testing centres, who were subsequently attending district centres for assessment for commencing ARVs. Patients (>18 years) were sampled randomly with no exclusion for gender or clinical criteria. Sequences were analysed according to phylogeny and drug resistance. RESULTS: Phylogenetic clustering within the cohort was suggestive of multiple introductions of subtype C virus into the region. Drug resistance mutations (according to the International AIDS Society-USA classification) were distributed randomly across the cohort phylogeny with an overall prevalence of 2.3% in the sampled patients. When stratified according to CD4(+) T-cell count, the prevalence of resistance was 3.6%, 0.9% and 1.2% for CD4(+) T-cell counts <100, 200-350 and >500 cells/microl, respectively, and was most common for non-nucleoside reverse transcriptase inhibitor resistance (3.1% in patients with CD4(+) T-cell count <100 cells/microl). We surveyed all drug-selected mutations and found further significant clustering among patients with low CD4(+) T-cell counts (P=0.003), suggesting unrecognized exposure to ARVs. CONCLUSIONS: In the Free State population, there was a statistical association between low CD4(+) T-cell counts and drug-associated viral polymorphisms. Our data advocate the benefit of detailed history taking from patients starting HAART at low CD4(+) T-cell counts with close follow-up of the virological response.

Salloum S, Oniangue-Ndza C, Neumann-Haefelin C, Hudson L, Giugliano S, aus dem Siepen M, Nattermann J, Spengler U et al. 2008. Escape from HLA-B*08-restricted CD8 T cells by hepatitis C virus is associated with fitness costs. J Virol, 82 (23), pp. 11803-11812. | Show Abstract | Read more

The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL(1395-1403) was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.

Nebbia G, Mattes FM, Smith C, Hainsworth E, Kopycinski J, Burroughs A, Griffiths PD, Klenerman P, Emery VC. 2008. Polyfunctional cytomegalovirus-specific CD4+ and pp65 CD8+ T cells protect against high-level replication after liver transplantation. Am J Transplant, 8 (12), pp. 2590-2599. | Show Abstract | Read more

To determine whether polyfunctional CD4+ T-cell responses coupled with CD8+ T-cell responses against human cytomegalovirus (HCMV) are key to the control of HCMV replication we prospectively analyzed 29 liver transplant recipients for CD4+ T-cell responses against soluble HCMV antigen, pp65 and IE1 proteins, CD8+ T-cell responses against pp65 and IE1 proteins and a range of T helper (Th) 1 and Th2 cytokines. Eleven patients (38%) developed HCMV DNAemia at a median of 21 days post-liver transplantation (range 17-31 days). There was a significantly lower frequency and absolute number of total HCMV CD4+ T cells producing IFNgamma, IFNgamma+IL2 and IL2 and pp65-CD8+ T cells producing IFNgamma in patients with DNAemia. The quantities of Th1 and Th2 cytokines present during the first 20 days posttransplant were not predictive of DNAemia. Cut-off levels during the first 20 days posttransplant of 0.1% of lysate stimulated CD4+ T cells producing IL2, and pp65-CD8+ T cells producing IFNgamma above 0.4% had positive and negative predictive values for DNAemia of 54% and 100% and 50% and 92%, respectively. Measuring polyfunctional CD4+ T cells against HCMV early posttransplant may allow targeted intervention to minimize the occurrence and acute and long-term consequences of HCMV replication.

Klenerman P, Dunbar PR. 2008. CMV and the art of memory maintenance. Immunity, 29 (4), pp. 520-522. | Show Abstract | Read more

The CD8(+) T cell responses to CMV gradually increase in magnitude over time-so-called memory "inflation." In this issue of Immunity, Snyder et al. (2008) examine the dynamics of memory inflation and demonstrate continuous turnover of inflating T cells, drawing on both memory cells and naive cells to replace them.

Gaudieri S, Rauch A, Pfafferott K, Barnes E, Cheng W, Mccaughan G, Shackel N, Jeffrey GP et al. 2008. Co-localisation of hepatitis C virus drug-resistant mutations and immune-driven adaptations for genotype 1 and 3: relevance to therapy outcome JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, 23 pp. A355-A355.

Ward SM, Phalora P, Bradshaw D, Leyendeckers H, Klenerman P. 2008. Direct ex vivo evaluation of long-lived protective antiviral memory B cell responses against hepatitis B virus. J Infect Dis, 198 (6), pp. 813-817. | Show Abstract | Read more

BACKGROUND: The frequency of protective antiviral memory B cells after hepatitis B virus (HBV) vaccination is unknown. METHODS: A novel 2-step immunomagnetic protocol to assess the ex vivo frequency of protective HBV surface antigen (HBsAg)-specific memory B cells was used. RESULTS: HBsAg-specific memory B cells were detected in vaccinated individuals, although at very low frequency (median, 0.2% of CD19(+) cells [range, 0%-4% of CD19(+) cells]). No correlation existed between the frequency of HBsAg-specific memory B cells and the corresponding serum antibody titer or B cell enzyme-linked immunosorbent spot findings. CONCLUSION: Our results indicate sustained B cell-mediated protection against HBV despite waning antibody titers, which is consistent with clinical observations.

Danta M, Semmo N, Fabris P, Brown D, Pybus OG, Sabin CA, Bhagani S, Emery VC, Dusheiko GM, Klenerman P. 2008. Impact of HIV on host-virus interactions during early hepatitis C virus infection. J Infect Dis, 197 (11), pp. 1558-1566. | Show Abstract | Read more

BACKGROUND: Human immunodeficiency virus (HIV) may influence the outcome and natural history of hepatitis C virus (HCV) infection through an impact on acute HCV-specific T cell responses. METHODS: Fifty-five HIV-positive males with acute HCV infection were identified; monoinfected individuals (n = 8) were used for peripheral blood mononuclear cell comparison. In 14 coinfected and 8 HCV-monoinfected patients, HCV-specific T cell responses against a range of HCV antigens were assessed using interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) and proliferation assays. E1/E2 region genetic diversity and the selection pressure on the virus were measured in 8 coinfected patients by use of cloned sequences over time. RESULTS: HCV persisted in 52 (95%) coinfected individuals. HCV/HIV coinfection significantly reduced IFN-gamma ELISpot responses versus those in HCV-monoinfected individuals, especially against nonstructural proteins (1/10 vs. 5/8; P = .008). In coinfected patients, increased HCV genetic diversity was observed between the first and subsequent time points, with no evidence for positive selection in the E1/E2 region sequenced. CONCLUSION: HIV coinfection is associated with increased rates of HCV persistence and a lack of critical CD4 T cell responses, with no evidence of immune selection pressure during early HCV infection. Loss of key cellular immune responses against HCV during acute disease may contribute to the failure of early host control of HCV in HCV/HIV-coinfected patients.

Strickland GT, El-Kamary SS, Klenerman P, Nicosia A. 2008. Hepatitis C vaccine: supply and demand. Lancet Infect Dis, 8 (6), pp. 379-386. | Show Abstract | Read more

Despite difficulties associated with extreme variability and mutability of hepatitis C virus (HCV), several vaccines that prevent initial infection or viral persistence, or that clear viraemia in individuals with chronic HCV infections, are currently in development. At least one vaccine that may prevent chronic persistent infections will soon be available for testing. We review the widespread importance of HCV infection and disease, the immune response to HCV and correlates of protection, prevention strategies and vaccine candidates, and groups that will need the vaccine and provide suitable populations for assessing vaccine safety and efficacy. The evaluation of prophylactic vaccines is particularly problematic since distribution must focus upon individuals at high risk of exposure-for example, intravenous drug users and health-care providers in areas with high HCV prevalence. Although there is a huge need for therapeutic vaccines, further immunological hurdles must be cleared before one becomes available.

Capraru D, Müller A, Csernok E, Gross WL, Holl-Ulrich K, Northfield J, Klenerman P, Herlyn K et al. 2008. Expansion of circulating NKG2D+ effector memory T-cells and expression of NKG2D-ligand MIC in granulomaous lesions in Wegener's granulomatosis. Clin Immunol, 127 (2), pp. 144-150. | Show Abstract | Read more

Expansion of circulating CD28- T-cells reminiscent of effector memory T-cells (T(EM)) has been reported in Wegener's granulomatosis (WG) recently. To investigate the role of T(EM) in WG, we analyzed the expression of the activating NK-receptor NKG2D and its ligand MIC on circulating T(EM) and in granulomatous lesions, respectively. NKG2D was anomalously expressed and preferentially detected on circulating CD4+CD28- T(EM) in WG. Compared to healthy controls, T(EM) display a more activated phenotype potentially favoring unbalanced proinflammatory responses in WG. Cluster-like formations of "Wegener's autoantigen" PR3 were surrounded by NKG2D+ and NKG2D-ligand MIC+ cells in WG-granulomata, but not in disease controls. Further, IL-15 - known to drive T(EM) differentiation and proliferation--was also expressed in WG-granulomata. Thus, through acquisition of NK-like "innate" properties, IL-15 stimulated NKG2D+ T(EM) could interact with MIC+ cells within WG-granulomata, thereby sustaining inflammation and autoimmunity and promoting self-perpetuating pathology in WG.

Mattes FM, Vargas A, Kopycinski J, Hainsworth EG, Sweny P, Nebbia G, Bazeos A, Lowdell M et al. 2008. Functional impairment of cytomegalovirus specific CD8 T cells predicts high-level replication after renal transplantation. Am J Transplant, 8 (5), pp. 990-999. | Show Abstract | Read more

Human cytomegalovirus (HCMV) remains an important cause of morbidity after allotransplantation, causing a range of direct effects including hepatitis, pneumonitis, enteritis and retinitis. A dominant risk factor for HCMV disease is high level viral replication in blood but it remains unexplained why only a subset of patients develop such diseases. In this detailed study of 25 renal transplant recipients, we show that functional impairment of HCMV specific CD8 T cells in the production of interferon gamma was associated with a 14-fold increased risk of progression to high level replication. The CD8 T-cell impairment persisted during the period of high level replication and was more prominent in patients above 40 years of age (odds ratio = 1.37, p = 0.01) and was also evident in dialysis patients. Threshold levels of functional impairment were associated with an increased risk of future HCMV replication and there was a direct relationship between the functional capacity of HCMV ppUL83 CD8 T cells and HCMV load (R(2)= 0.83). These results help to explain why a subset of seropositive individuals develop HCMV replication and are at risk of end-organ disease and may facilitate the early identification of individuals who would benefit from targeted anti-HCMV therapy after renal transplantation.

Neumann-Haefelin C, Frick DN, Wang JJ, Pybus OG, Salloum S, Narula GS, Eckart A, Biezynski A et al. 2008. Analysis of the evolutionary forces in an immunodominant CD8 epitope in hepatitis C virus at a population level. J Virol, 82 (7), pp. 3438-3451. | Show Abstract | Read more

Failure of the adaptive immune response to control infection with the hepatitis C virus (HCV) can result from mutational escape in targeted T-cell epitopes. Recent studies suggest that T-cell immune pressure is an important factor in the evolution of the nonstructural proteins in HCV. The aim of this study was to characterize the forces that contribute to viral evolution in an HLA-A*01-restricted epitope in HCV NS3. This epitope represents a potentially attractive target for vaccination strategies since it is conserved across all genotypes. In our cohort of subjects with chronic HCV infection (genotype 1b or 3a), it is a frequently recognized CD8 epitope in HLA-A*01-positive subjects. Viral sequence data reveal that an escape variant is the dominant residue in both genotypes. The predominant Y1444F substitution seemingly impairs binding to the HLA-A*01 molecule, which may have an important impact on the ability to prime a functional CD8 response upon infection. Interestingly, a case of evolution toward the prototype sequence was observed during chronic infection, possibly because the helicase activity of the protein containing the Y1444F substitution is reduced compared to the prototype sequence. Comparison of HCV sequences from Asia and Europe suggests that the frequency of the HLA-A*01 allele in a population may influence the frequency of the escape variant in circulating strains. These data suggest a complex interaction of multiple forces shaping the evolution of HCV in which immune pressure both within the individual and also at the population level in addition to functional constraints are important contributing factors.

Kasprowicz V, Schulze Zur Wiesch J, Kuntzen T, Nolan BE, Longworth S, Berical A, Blum J, McMahon C et al. 2008. High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome. J Virol, 82 (6), pp. 3154-3160. | Show Abstract | Read more

We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8(+) and CD4(+) T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.

Barnes E, Salio M, Cerundolo V, Francesco L, Pardoll D, Klenerman P, Cox A. 2008. Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus. J Viral Hepat, 15 (3), pp. 219-228. | Show Abstract | Read more

Studies assessing the function of monocyte derived dendritic cells (MD-DC) in individuals with hepatitis C virus (HCV) infection have shown conflicting results. Impaired MD-DC function in chronic HCV infection would have important implications both for understanding the pathogenesis of HCV infection and in the use of autologous MD-DC in vaccination strategies. We determined the allostimulatory capacity of MD-DC in the same patient before and after HCV infection. Next, the phenotype, cytokine production and allostimulatory function of immature and mature MD-DC in individuals with persistent HCV infection were compared directly with MD-DC from healthy individuals. Finally, we assessed the ability of MD-DC to prime autologous naïve peptide specific CD8+ T cells using HLA-A2 class-I tetramers. DCs retained the same allostimulatory capacity before and following the establishment of persistent HCV infection. The surface phenotype and the amount of interleukin (IL)-10 and IL-12(p70) produced during DC maturation did not differ between HCV-infected individuals and healthy controls. Mature DCs from HCV-infected individuals performed comparably in an allogeneic MLR compared with healthy individuals. Mature MD-DC from HCV-infected individuals stimulated the expansion of peptide specific naïve CD8+ T cells. MD-DC from HCV-infected and healthy individuals are phenotypically indistinguishable and perform comparably in functional assays.

Kasprowicz V, Ward SM, Turner A, Grammatikos A, Nolan BE, Lewis-Ximenez L, Sharp C, Woodruff J et al. 2008. Defining the directionality and quality of influenza virus-specific CD8+ T cell cross-reactivity in individuals infected with hepatitis C virus. J Clin Invest, 118 (3), pp. 1143-1153. | Show Abstract | Read more

Cross-reactivity of murine and recently human CD8(+) T cells between different viral peptides, i.e., heterologous immunity, has been well characterized. However, the directionality and quality of these cross-reactions is critical in determining their biological importance. Herein we analyzed the response of human CD8(+) T cells that recognize both a hepatitis C virus peptide (HCV-NS3) and a peptide derived from the influenza neuraminidase protein (Flu-NA). To detect the cross-reactive CD8(+) T cells, we used peptide-MHC class I complexes (pMHCs) containing a new mutant form of MHC class I able to bind CD8 more strongly than normal MHC class I complexes. T cell responses against HCV-NS3 and Flu-NA peptide were undetectable in normal donors. In contrast, some responses against the Flu-NA peptide were identified in HCV(+) donors who showed strong HCV-NS3-specific reactivity. The Flu-NA peptide was a weak agonist for CD8(+) T cells in HCV(+) individuals on the basis of novel pMHCs and functional assays. These data support the idea of cross-reactivity between the 2 peptides, but indicate that reactivity toward the Flu-NA peptide is highly CD8-dependent and occurs predominantly after priming during HCV infection. Our findings indicate the utility of the novel pMHCs in dissecting cross-reactivity and suggest that cross-reactivity between HCV and influenza is relatively weak. Further studies are needed to relate affinity and functionality of cross-reactive T cells.

Hughes D, Hafferty J, Fulton L, Friend P, Devaney A, Loke J, Welsh KI, Handa A, Klenerman P. 2008. Donor and recipient CMV serostatus and antigenemia after renal transplantation: an analysis of 486 patients. J Clin Virol, 41 (2), pp. 92-95. | Show Abstract | Read more

BACKGROUND: Cytomegalovirus infection in renal transplant recipients is a major clinical problem, with both short and long term sequelae. Infection can occur as a result of reactivation of latent virus or new infection from donor tissues. The impact of donor and recipient serostatus on viremia is well recognised, with seronegative recipients at greatest risk after transplantation of an organ from a seropositive donor. However, the impact of grafting such organs into seropositive recipients is less clear. OBJECTIVES: To assess the impact of recipient serostatus on the risk of CMV antigenemia in a large renal transplant cohort. STUDY DESIGN: We prospectively quantified CMV antigenemia over time in a cohort of 486 recipients. We analysed the antigenemia status according to donor and recipient serostatus. RESULTS: Antigenemia was most common in seronegative recipients of organs from seropositive donors (D+/R-). Nevertheless, we observed that even in CMV seropositive recipients, the impact of donor serostatus on CMV antigenemia is still substantial (p=0.006; OR=2.2). CONCLUSIONS: In this large study, donor serostatus clearly plays a significant role in determining CMV risk, even in seropositive recipients.

Northfield JW, Kasprowicz V, Lucas M, Kersting N, Bengsch B, Kim A, Phillips RE, Walker BD, Thimme R, Lauer G, Klenerman P. 2008. CD161 expression on hepatitis C virus-specific CD8+ T cells suggests a distinct pathway of T cell differentiation. Hepatology, 47 (2), pp. 396-406. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) causes chronic infection accompanied by a high risk of liver failure and hepatocellular carcinoma. CD8+ T cell responses are important in the control of viremia. However, the T cell response in chronic infection is weak both in absolute numbers and in the range of epitopes targeted. In order to explore the biology of this response further, we analyzed expression of a panel of natural killer cell markers in HCV compared with other virus-specific T cell populations as defined by major histocompatibility complex class I tetramers. We found that CD161 was significantly expressed on HCV-specific cells (median 16.8%) but not on CD8+ T cells specific for human immunodeficiency virus (3.3%), cytomegalovirus (3.4%), or influenza (3.4%). Expression was seen in acute, chronic, and resolved disease and was greatest on intrahepatic HCV-specific T cells (median 57.6%; P < 0.05). Expression of CD161 was also found on hepatitis B virus-specific CD8+ T cells. In general, CD161+CD8+ T cells were found to be CCR7- "effector memory" T cells that could produce proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) but contained scanty amounts of cytolytic molecules (granzyme B and perforin) and proliferated poorly in vitro. Expression of CD161 on CD8+ T cells was tightly linked to that of CXCR6, a chemokine with a major role in liver homing. CONCLUSION: We propose that expression of CD161 indicates a unique pattern of T cell differentiation that might help elucidate the mechanisms of HCV immunity and pathogenesis.

Fitzmaurice K, Klenerman P. 2008. Cellular immunity and acute hepatitis C infection. Curr Pharm Des, 14 (17), pp. 1666-1677. | Show Abstract | Read more

Hepatitis C virus (HCV) infects an estimated 170 million people globally and persistent infection within the liver is the usual outcome of infection. The resulting liver disease leads to substantial morbidity and to date no vaccine exists. Furthermore the treatments available are frequently ineffective. A minority of those exposed will however successfully control the virus and the factors that dictate this remain elusive. The events that occur in the immediate and early phase post exposure are thought to play a crucial role in determining the outcome and virus specific T cells have a confirmed role in directing the immune response towards a successful outcome. An understanding of the T cell responses and the strategies, which allow the virus to evade these responses in the majority, is an essential prerequisite both for vaccine design and the development of therapeutic agents. We review here the characteristics of the cellular immune responses in acute infection and how the virus manages to undermine these responses and establish chronicity.

Klenerman P, Barnes E. 2008. Immune responses against the hepatitis C virus and the outcome of therapy pp. 71-86. | Show Abstract | Read more

The hepatitis C virus (HCV) sets up persistence in the majority of those infected. In doing so, it evades both innate and adaptive immune responses. However, in a reasonable fraction of patients (20-50%), there is long-term control of viremia through some effective combination of host responses. It is generally considered that cellular immune responses-mediated by CD4+ and CD8+ T cells-play a major role in determining this successful outcome, although they do so in concert with many other cellular and humoral mediators (Ward et al., 2002). © 2008 Springer Science+Business Media, LLC.

Hennig BJW, Hellier S, Frodsham AJ, Zhang L, Klenerman P, Knapp S, Wright M, Thomas HC, Thursz M, Hill AVS. 2007. Association of low-density lipoprotein receptor polymorphisms and outcome of hepatitis C infection GENES AND IMMUNITY, 8 (8), pp. 707-707. | Read more

Klenerman P, Kim A. 2007. HCV-HIV coinfection: simple messages from a complex disease. PLoS Med, 4 (10), pp. e240. | Read more

Semmo N, Klenerman P. 2007. CD4+ T cell responses in hepatitis C virus infection. World J Gastroenterol, 13 (36), pp. 4831-4838. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are relatively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type I profile, which promotes cellular effector mechanisms are thought to be more likely to experience viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells, especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in determining the outcome of acute and chronic HCV infection will be discussed in this review.

Ward SM, Fox BC, Brown PJ, Worthington J, Fox SB, Chapman RW, Fleming KA, Banham AH, Klenerman P. 2007. Quantification and localisation of FOXP3+ T lymphocytes and relation to hepatic inflammation during chronic HCV infection. J Hepatol, 47 (3), pp. 316-324. | Show Abstract | Read more

BACKGROUND/AIMS: T lymphocyte-mediated immune reactions are closely involved in the pathogenesis of HCV-induced chronic liver disease. Regulatory T cells are able to suppress HCV-specific T lymphocyte proliferation and cytokine secretion during chronic HCV infection. We wished to address to what extent regulatory T cells exist in the livers of HCV+ individuals, and what the role of such cells might be in disease progression. METHODS: We analysed the frequency and distribution of FOXP3+ cells, along with CD4, CD8 and CD20+ cells, in liver biopsies of 28 patients with chronic HCV and 14 patients with PBC, and correlated these data with histological parameters. RESULTS: A striking number of FOXP3+ cells were present in the portal tract infiltrates of HCV-infected livers. FOXP3+ cells were largely CD4+ and there was a remarkably consistent ratio of total CD4+:FOXP3+ cells in liver across a wide range of disease states of around 2:1. This differed substantially from the ratio observed in PBC (10:1, P=0.001). CONCLUSIONS: An unexpectedly high proportion of the cellular infiltrate in persistent HCV infection comprises FOXP3+ cells. The relative proportion of FOXP3+ cells remains similar in both mild and severe fibrosis. These cells are likely to play a critical role in intrahepatic immune regulation, although their overall role in disease progression remains to be determined.

Semmo N, Krashias G, Willberg C, Klenerman P. 2007. Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection. J Viral Hepat, 14 (7), pp. 492-502. | Show Abstract | Read more

CD4(+) T-cell responses are important for the outcome of hepatitis C virus (HCV) infection. However, the functional status of HCV-specific CD4(+) T cells in persistent infection is poorly understood. It is generally recognized that proliferative capacity of HCV-specific CD4(+) T cells is weak or absent in persistent infection, but whether this results from deletion of antigen-specific cells or represents maintenance of antigen-specific but poorly proliferative populations is not defined. We used a set of ex vivo assays to evaluate the functionality of HCV specific CD4(+) T cells in persistent and resolved infection. Peripheral blood mononuclear cells (PBMC) from 24 prospectively recruited HCV polymerase chain reaction (PCR) positive individuals, 12 spontaneously resolved individuals (i.e. anti-HCV+, PCR-) and 11 healthy controls were analysed for interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) secretion by enzyme linked immunospot assays (ELISpot). HCV-specific CD4(+) proliferative responses of carboxy fluorescein succinimidyl ester-labelled PBMC were assessed using a sensitive single cell flow cytometric assay. Sustained IFN-gamma ELISpot responses were observed in the PCR+ group. However, proliferation of HCV-specific CD4(+) T cells in the PCR+ group was substantially reduced on a per cell basis, in parallel to IL-2 secretion, compared with responses in the PCR- group. In PCR- individuals, a strong relationship between cytokine secretion and proliferative capacity was seen. However, in PCR+ individuals, IFN-gamma secretion far exceeded proliferative capacity. During persistent HCV infection, some CD4(+) T-cell specificities appear to be lost, as measured using a range of techniques, but others, potentially important, are maintained as IFN-gamma secretors but with low proliferative capacity even using a highly sensitive assay. Such subsets may yet play a significant role in vivo and also provide a template for modulation in immunotherapeutic interventions.

Wodarz D, Sierro S, Klenerman P. 2007. Dynamics of killer T cell inflation in viral infections. J R Soc Interface, 4 (14), pp. 533-543. | Show Abstract | Read more

Upon acute viral infection, a typical cytotoxic T lymphocyte (CTL) response is characterized by a phase of expansion and contraction after which it settles at a relatively stable memory level. Recently, experimental data from mice infected with murine cytomegalovirus (MCMV) showed different and unusual dynamics. After acute infection had resolved, some antigen specific CTL started to expand over time despite the fact that no replicative virus was detectable. This phenomenon has been termed as "CTL memory inflation". In order to examine the dynamics of this system further, we developed a mathematical model analysing the impact of innate and adaptive immune responses. According to this model, a potentially important contributor to CTL inflation is competition between the specific CTL response and an innate natural killer (NK) cell response. Inflation occurs most readily if the NK cell response is more efficient than the CTL at reducing virus load during acute infection, but thereafter maintains a chronic virus load which is sufficient to induce CTL proliferation. The model further suggests that weaker NK cell mediated protection can correlate with more pronounced CTL inflation dynamics over time. We present experimental data from mice infected with MCMV which are consistent with the theoretical predictions. This model provides valuable information and may help to explain the inflation of CMV specific CD8+T cells seen in humans as they age.

Chuang WC, Sarkodie F, Brown CJ, Owusu-Ofori S, Brown J, Li C, Navarrete C, Klenerman P, Allain JP. 2007. Protective effect of HLA-B57 on HCV genotype 2 infection in a West African population. J Med Virol, 79 (6), pp. 724-733. | Show Abstract | Read more

Recovery from Hepatitis C virus (HCV) infection is considered infrequent (<20%) in western populations but reaches 50% in West Africa where genotype 2 infection is predominant. To investigate the role of cellular immune responses and host genetics in this phenomenon, samples from 104 Ghanaian blood donors reactive with anti-HCV assays were collected between 2000 and 2005. HCV antibody was confirmed by Western blot using genotype 2 recombinant core, E2 and NS3 proteins. Viral load and genotype were determined. Samples were stratified into 37 chronic, 35 recovered infections and 32 false positive. Eighty-one percentage of subjects with chronic infection (RNA positive) carried genotype 2 HCV. Cellular immune response was investigated in 35 frozen peripheral blood mononuclear cell (PBMC) samples suitable for interferon-gamma ELISPOT assay. Twelve out of 24 confirmed recovered, 1 out of 5 chronically infected and none of the 6 false-positive controls reacted to recombinant proteins. HLA-A, -B and -DR types were determined by DNA methodology. HLA-B*57 was significantly more frequent in the group which had recovered from HCV infection compared with chronically infected subjects (P = 0.0053, OR = 8.02). In conclusion, it is hypothesized that the dominance of genotype 2 HCV strains may be an important factor explaining the high rate of recovery from HCV infections in Ghana via an efficient contribution of HLA-B*57 which is relatively frequent in the population.

Northfield JW, Loo CP, Barbour JD, Spotts G, Hecht FM, Klenerman P, Nixon DF, Michaëlsson J. 2007. Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point. J Virol, 81 (11), pp. 5759-5765. | Show Abstract | Read more

CD8(+) T cells are believed to play an important role in the control of human immunodeficiency virus type 1 (HIV-1) infection. However, despite intensive efforts, it has not been possible to consistently link the overall magnitude of the CD8(+) T-cell response with control of HIV-1. Here, we have investigated the association of different CD8(+) memory T-cell subsets responding to HIV-1 in early infection with future control of HIV-1 viremia. Our results demonstrate that both a larger proportion and an absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(+) effector memory T cells (T(EMRA) cells) were associated with a lower future viral load set point. In contrast, a larger absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(-) effector memory T cells (T(EM)) was not related to the viral load set point. Overall, the findings suggest that CD8(+) T(EMRA) cells have superior antiviral activity and indicate that both qualitative and quantitative aspects of the CD8(+) T-cell response need to be considered when defining the characteristics of protective immunity to HIV-1.

Millington KA, Innes JA, Hackforth S, Hinks TS, Deeks JJ, Dosanjh DP, Guyot-Revol V, Gunatheesan R, Klenerman P, Lalvani A. 2007. Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load. J Immunol, 178 (8), pp. 5217-5226. | Show Abstract

Distinct IFN-gamma and IL-2 profiles of Ag-specific CD4(+) T cells have recently been associated with different clinical disease states and Ag loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis Ag-specific T cells secreting IFN-gamma and IL-2 in 23 patients with untreated active tuberculosis when bacterial and Ag loads are high and after curative treatment, when Ag load is reduced. The frequencies of M. tuberculosis Ag-specific IFN-gamma-secreting T cells declined during 28 mo of follow-up with an average percentage decline of 5.8% per year (p = 0.005), while the frequencies of Ag-specific IL-2-secreting T cells increased during treatment (p = 0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-gamma- and IL-2-secreting cells over 28 mo. Simultaneous measurement of IFN-gamma and IL-2 secretion at the single-cell level revealed a codominance of IFN-gamma-only secreting and IFN-gamma/IL-2 dual secreting CD4(+) T cells in active disease that shifted to dominance of IFN-gamma/IL-2-secreting CD4(+) T cells and newly detectable IL-2-only secreting CD4(+) T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis that now requires validation in larger prospective studies.

Klenerman P, McMichael A. 2007. AIDS/HIV. Finding footprints among the trees. Science, 315 (5818), pp. 1505-1507. | Show Abstract | Read more

High-quality statistical analyses allow tracking of HIV evasion of the host immune response through epitope mutations and suggest refined strategies for vaccine development.

Lucas M, Ulsenheimer A, Pfafferot K, Heeg MH, Gaudieri S, Grüner N, Rauch A, Gerlach JT et al. 2007. Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection. PLoS One, 2 (7), pp. e649. | Show Abstract | Read more

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Willberg CB, Ward SM, Clayton RF, Naoumov NV, McCormick C, Proto S, Harris M, Patel AH, Klenerman P. 2007. Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha. PLoS One, 2 (8), pp. e791. | Show Abstract | Read more

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

Day CL, Kiepiela P, Leslie AJ, van der Stok M, Nair K, Ismail N, Honeyborne I, Crawford H et al. 2007. Proliferative capacity of epitope-specific CD8 T-cell responses is inversely related to viral load in chronic human immunodeficiency virus type 1 infection. J Virol, 81 (1), pp. 434-438. | Show Abstract | Read more

The relationship between the function of human immunodeficiency virus (HIV)-specific CD8 T-cell responses and viral load has not been defined. In this study, we used a panel of major histocompatibility complex class I tetramers to examine responses to frequently targeted CD8 T-cell epitopes in a large cohort of antiretroviral-therapy-naïve HIV type 1 clade C virus-infected persons in KwaZulu Natal, South Africa. In terms of effector functions of proliferation, cytokine production, and degranulation, only proliferation showed a significant correlation with viral load. This robust inverse relationship provides an important functional correlate of viral control relevant to both vaccine design and evaluation.

Hennig BJW, Hellier S, Frodsham AJ, Zhang L, Klenerman P, Knapp S, Wright M, Thomas HC, Thursz M, Hill AVS. 2007. Erratum: Association of low-density lipoprotein receptor polymorphisms and outcome of hepatitis C infection (Genes and Immunity (2002) vol. 3 (359-367) 10.1038/sj.gene.6363883) Genes and Immunity, 8 (8), pp. 707. | Read more

Komatsu H, Kogawa K, Nonoyama S, Inui A, Sogo T, Fujisawa T, Klenerman P. 2006. Bone marrow transplantation from a pediatric donor with a high frequency of cytomegalovirus-specific T-cells. J Med Virol, 78 (12), pp. 1616-1623. | Show Abstract | Read more

A recent study reported that quantitation of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in the graft and monitoring of these T cells might identify hematopoietic stem cell transplantation-recipients at the risk for progressive CMV infection. A 6-year-old girl underwent bone marrow transplantation from an HLA-identical sibling with a very high frequency of CMV specific tetramer-positive CD8+ T-cells. CMV-specific T-cell immunity was prospectively evaluated using a peptide (HLA-A2, NLVPMVATV). Tetramer assay showed that the frequency of CMV-specific CD8+ T cells of the donor in the peripheral blood was 5.3%, higher than average amongst young children. The frequency of CMV-specific CD8+ T cells of the donor in the graft was 3.7% of CD8+ T-cells. Before transplantation, the frequency of CMV specific CD8+ T cells of the recipient was 0.1% in the peripheral blood. Surprisingly, the frequency of CMV specific CD8+ T cells increased up to 30% of CD8+ T-cells at day 27 after transplantation. IFN-gamma enzyme-linked immunospot assay showed the recipient-T cells had strong responses to the A2-specific NLVPMVATV peptide. Although the phenotypic pattern of the CMV-specific T cells of the recipient was different from those of the donor before transplantation, the phenotype of the donor-derived cells retained their original phenotype in the recipient after transplantation. These finding suggested that active transferred immunity from the graft with a high frequency of CMV-specific CTL could induce a rapid reconstitution of CMV-specific T-cell mediated immunity in pediatric HLA-identical allogenetic bone marrow transplantation. The screening of peripheral blood using HLA-peptide tetramer staining might be beneficial to select donors.

Klenerman P, Ludewig B. 2006. Virus scores a perfect 10. Nat Med, 12 (11), pp. 1246-1248. | Show Abstract | Read more

Blockade of a single cytokine - interleukin-10 - can re-energize T cells 'exhausted' by persistent virus infection. The findings have implications for controlling HIV and other persistent viruses. © 2006 Nature Publishing Group.

Kasprowicz V, Isa A, Tolfvenstam T, Jeffery K, Bowness P, Klenerman P. 2006. Tracking of peptide-specific CD4+ T-cell responses after an acute resolving viral infection: a study of parvovirus B19. J Virol, 80 (22), pp. 11209-11217. | Show Abstract | Read more

The evolution of peptide-specific CD4(+) T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4(+) T-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared to those for a cohort of B19-specific immunoglobulin M-negative (IgM(-)), IgG(+) remotely infected individuals. Both cohorts of individuals were found to make broad CD4(+) responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals were only detected after culture. One epitope (LASEESAFYVLEHSSFQLLG) was consistently targeted by both acutely (10/12) and remotely (6/7) infected individuals. This epitope was DRB1*1501 restricted, and a major histocompatibility complex peptide tetramer stained PBMCs from acutely infected individuals in the range of 0.003 to 0.042% of CD4(+) T cells. Tetramer-positive populations were initially CD62L(lo); unlike the case for B19-specific CD8(+) T-cell responses, however, CD62L was reexpressed at later times, as responses remained stable or declined slowly. This first identification of B19 CD4(+) T-cell epitopes, including a key immunodominant peptide, provides the tools to investigate the breadth, frequency, and functions of cellular responses to this virus in a range of specific clinical settings and gives an important reference point for analysis of peptide-specific CD4(+) T cells during acute and persistent virus infections of humans.

Harcourt GC, Scriba TJ, Semmo N, Bounds S, Taylor E, Klenerman P. 2006. Identification of key peptide-specific CD4+ T cell responses to human cytomegalovirus: implications for tracking antiviral populations. Clin Exp Immunol, 146 (2), pp. 203-210. | Show Abstract | Read more

Human cytomegalovirus (CMV) infection is normally controlled effectively by the immune response, including CD4(+) T cells. Large numbers of these cells are present in healthy seropositive individuals but their loss in immunosuppression leads to reactivation and disease. Tracking such responses in vivo is hampered by poor definition of their peptide targets. In this study, we defined the key targets of the peptide-specific CD4(+) T cell responses to the CMV pp65 protein using functional assays and a peptide library. Despite a good deal of interindividual variation in the numbers of peptides recognized, responses to CMV pp65 were strikingly targeted at three key epitopes. A response to one or more of these three key peptides was seen in all individuals tested (P < 0.0001) and this finding was tested and reproduced in a second independent population. The most common response identified was that to a DR53 restricted epitope, aa281-295. HLA-DR1 restricted CMV pp65-specific populations, although reproducibly detected, were of low frequency ex vivo. However, it was possible to detect and phenotype these cells using an enrichment protocol and this revealed them to have 'effector memory' status although, in contrast to CD8(+) T cell responses, these were CD45RA(-). These data suggest that CD4(+) T cell responses to CMV can be identified reliably using a pool of just three peptides. This simple approach will provide a robust and reliable as well as economic method for tracking peptide specific populations in health and disease.

Ulsenheimer A, Lucas M, Seth NP, Tilman Gerlach J, Gruener NH, Loughry A, Pape GR, Wucherpfennig KW, Diepolder HM, Klenerman P. 2006. Transient immunological control during acute hepatitis C virus infection: ex vivo analysis of helper T-cell responses. J Viral Hepat, 13 (10), pp. 708-714. | Show Abstract | Read more

Hepatitis C virus (HCV) readily sets up persistence after acute infection. Cellular immune responses are thought to play a major role in control of the virus. Failure of CD4+ T-cell responses in acute disease is associated with viral persistence but the dynamics of this are poorly understood. We aimed to assess such responses using a novel set of Class II tetrameric complexes (tetramers) to study helper T-cells ex vivo in acute disease. We analysed the HCV-specific CD4+ T-cell response in a patient with acute hepatitis c infection. We were able to track the virus-specific CD4+ T-cells directly ex vivo with HLA DR4 tetramers. Proliferative responses were absent initially, recovered as viral load dropped and were lost again during relapse. Longitudinal tetramer analyses showed expanded populations of antiviral CD4+ T-cells throughout acute infection despite lack of proliferation. A pattern of transient CD4+ T-cell proliferative responses as HCV is partially controlled is observed. Failure to control virus is associated with emergence of 'dysfunctional' CD4+ T-cell populations. Failure to control HCV in acute disease may relate to the capacity to sustain efficient immune responses as virus attempts to 'bounce back' after partial control.

Harcourt G, Gomperts E, Donfield S, Klenerman P. 2006. Diminished frequency of hepatitis C virus specific interferon gamma secreting CD4+ T cells in human immunodeficiency virus/hepatitis C virus coinfected patients. Gut, 55 (10), pp. 1484-1487. | Show Abstract | Read more

BACKGROUND: Human immunodeficiency virus/hepatitis C virus (HIV/HCV) coinfection is a common and complex clinical problem in which loss of immunological control of HCV occurs, with increased HCV viral load and more aggressive liver disease. Cellular immune responses, particularly secretion of interferon gamma (IFN-gamma) appear to be important in the control of HCV, and a detectable HCV specific CD4 response is associated with clearance of the virus. HCV specific CD8+ T cell responses, weak in chronic HCV infection, have been shown to be further impaired in HIV coinfection and this CD8+ T cell deficiency is related to the decline in CD4 T cell count. AIMS: To compare the CD4 T cell response to HCV in HIV/HCV coinfected and HCV monoinfected individuals and to determine the relationship of responses with declining CD4 count. PATIENTS: The study subjects were a cohort of 68 HCV monoinfected and 67 HCV/HIV coinfected haemophiliac children and adolescents (the Hemophilia Growth and Development Study) who were followed for a seven year period. METHODS: We analysed IFN-gamma secreting CD4+ responses to HCV proteins and peptides and HIV p24 antigen using an ELISpot assay. RESULTS: We found a significant decrease in HCV specific responses among those who were HIV coinfected (10/67 v 36/68; p<0.0001) both in numbers of responders and frequency of specific cells. This did not appear to be closely related to CD4 count. CONCLUSIONS: The reduction in HCV specific CD4 T cells in coinfection provide a cellular mechanism for the loss of control of HCV in coinfected individuals, even in those with relatively preserved CD4+ T cell counts and CD4+ T cell responses to HIV.

Wherry EJ, Day CL, Draenert R, Miller JD, Kiepiela P, Woodberry T, Brander C, Addo M, Klenerman P, Ahmed R, Walker BD. 2006. HIV-specific CD8 T cells express low levels of IL-7Ralpha: implications for HIV-specific T cell memory. Virology, 353 (2), pp. 366-373. | Show Abstract | Read more

Chronic infections in mice can result in defects in memory CD8 T cell properties including low expression of the IL-7Ralpha (CD127). To determine whether defects in memory CD8 T cell formation exist during human chronic infections and to what extent these defects may be allele- or epitope-specific, we compared influenza (Flu), vaccinia (VV) and EBV-specific CD8 T cells to HIV-specific CD8 T cells, using a panel of 13 HIV tetramers. Compared to Flu, VV or EBV, HIV tetramer+ CD8 T cells expressed significantly lower levels of CD127, and this reduction was pervasive across all epitopes and alleles tested and over a wide range of viral loads and CD4 counts. These results indicate impaired HIV-specific memory CD8 T cell differentiation, regardless of level of control of viremia, epitopes targeted or restricting HLA alleles.

Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD et al. 2006. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature, 443 (7109), pp. 350-354. | Show Abstract | Read more

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.

Valdar W, Solberg LC, Gauguier D, Burnett S, Klenerman P, Cookson WO, Taylor MS, Rawlins JN, Mott R, Flint J. 2006. Genome-wide genetic association of complex traits in heterogeneous stock mice. Nat Genet, 38 (8), pp. 879-887. | Show Abstract | Read more

Difficulties in fine-mapping quantitative trait loci (QTLs) are a major impediment to progress in the molecular dissection of complex traits in mice. Here we show that genome-wide high-resolution mapping of multiple phenotypes can be achieved using a stock of genetically heterogeneous mice. We developed a conservative and robust bootstrap analysis to map 843 QTLs with an average 95% confidence interval of 2.8 Mb. The QTLs contribute to variation in 97 traits, including models of human disease (asthma, type 2 diabetes mellitus, obesity and anxiety) as well as immunological, biochemical and hematological phenotypes. The genetic architecture of almost all phenotypes was complex, with many loci each contributing a small proportion to the total variance. Our data set, freely available at http://gscan.well.ox.ac.uk, provides an entry point to the functional characterization of genes involved in many complex traits.

Munks MW, Cho KS, Pinto AK, Sierro S, Klenerman P, Hill AB. 2006. Four distinct patterns of memory CD8 T cell responses to chronic murine cytomegalovirus infection. J Immunol, 177 (1), pp. 450-458. | Show Abstract

CMVs are beta herpesviruses that establish lifelong latent infection of their hosts. Acute infection of C57BL/6 mice with murine CMV elicits a very broad CD8 T cell response, comprising at least 24 epitopes from 18 viral proteins. In contrast, we show here that the CD8 T cell response in chronically infected mice was dominated by only five epitopes. Altogether, four distinct CD8 T cell kinetic patterns were evident. Responses to some epitopes, including M45, which dominates the acute response, contracted sharply after day 7 and developed into stable long-term memory. The response to m139 underwent rapid expansion and contraction, followed by a phase of memory inflation, whereas the response to an M38 epitope did not display any contraction phase. Finally, responses against two epitopes encoded by the immediate early gene IE3 were readily detectable in chronically infected mice but near the limit of detection during acute infection. CD8 T cells specific for the noninflationary M45 epitope displayed a classic central memory phenotype, re-expressing the lymph node homing receptor CD62L and homeostatic cytokine receptors for IL-7 and IL-15, and produced low levels of IL-2. Responses to two inflationary epitopes, m139 and IE3, retained an effector memory surface phenotype (CD62L(low), IL-7Ralpha(-), IL-15Rbeta(-)) and were unable to produce IL-2. We suggest that immunological choices are superimposed on altered viral gene expression profiles to determine immunodominance during chronic murine CMV infection.

Kasprowicz V, Isa A, Jeffery K, Broliden K, Tolfvenstam T, Klenerman P, Bowness P. 2006. A highly restricted T-cell receptor dominates the CD8+ T-cell response to parvovirus B19 infection in HLA-A*2402-positive individuals. J Virol, 80 (13), pp. 6697-6701. | Show Abstract | Read more

Six of seven HLA-A*2402-positive individuals with acute parvovirus B19 infections made vigorous CD8-positive cytotoxic T-cell (CTL) responses to the viral epitope FYTPLADQF. All responders showed highly focused T-cell receptor (TCR) usage, using almost exclusively BV5.1. The BV5.1 TCR dominated the acute response, was maintained over time, and was also used by a remotely infected individual. Nine CTL clones and two oligoclonal lines obtained from three unrelated individuals used BV5.1, BJ2.1, and a conserved TCR CDR3 of nine amino acids. This commonly recognized epitope is likely important in long-term protective immunity and should be included in vaccine design.

Klenerman P, Semmo N. 2006. Cellular immune responses against persistent hepatitis C virus: gone but not forgotten. Gut, 55 (7), pp. 914-916. | Read more

Semmo N, Lucas M, Krashias G, Lauer G, Chapel H, Klenerman P. 2006. Maintenance of HCV-specific T-cell responses in antibody-deficient patients a decade after early therapy. Blood, 107 (11), pp. 4570-4571. | Read more

Goulder P, Klenerman P. 2006. Cytotoxic T lymphocytes and viral adaptation in HIV infection. Curr Opin HIV AIDS, 1 (3), pp. 241-248. | Show Abstract | Read more

PURPOSE OF REVIEW: Cytotoxic T lymphocytes are central in determining the extent of immune control of HIV infection. We examine the degree to which one may trace the steps that underlie these human leucocyte antigen/disease outcome associations. These findings will be of relevance to HIV vaccine design. RECENT FINDINGS: Initial investigations suggested that immune escape was inevitably associated with loss of control of viral replication. Recent studies of successful immune control of HIV infection, however, indicate that immune selection pressure on the virus may in certain circumstances result in viral adaptation with a reduced replication capacity. The dominant influence of particular human leucocyte antigen B alleles on HIV disease outcome is echoed in hepatitis C infection, and major histocompatibility complex class I alleles in the non-human primate that are associated with control of simian immunodeficiency virus infection also share similar mechanisms of viral control. SUMMARY: Cytotoxic T lymphocyte-mediated selection pressure on HIV has important consequences for subsequent immune control within the host. The extent to which viral adaptation occurs in the general population to alter these consequences is complex and epitope-specific, but is critical to the ongoing effectiveness of particular cytotoxic T lymphocyte responses through the course of the epidemic.

Neumann-Haefelin C, McKiernan S, Ward S, Viazov S, Spangenberg HC, Killinger T, Baumert TF, Nazarova N et al. 2006. Dominant influence of an HLA-B27 restricted CD8+ T cell response in mediating HCV clearance and evolution. Hepatology, 43 (3), pp. 563-572. | Show Abstract | Read more

Virus-specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well-defined cohort of women inoculated with HCV from a single source showed that HLA-B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA-B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA-B27 restricted hepatitis C virus (HCV)-specific CD8+ T cell epitope that is recognized in the majority of recovered HLA-B27 positive women. In chronically HCV-infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA-B27, indicating allele-specific selection pressure at the population level. Functional analysis in 3 chronically HCV-infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA-B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus-specific CD8+ T cells and the genetically determined restriction of the virus-specific T cell repertoire in HCV infection.

Solberg LC, Valdar W, Gauguier D, Nunez G, Taylor A, Burnett S, Arboledas-Hita C, Hernandez-Pliego P et al. 2006. A protocol for high-throughput phenotyping, suitable for quantitative trait analysis in mice. Mamm Genome, 17 (2), pp. 129-146. | Show Abstract | Read more

Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.

Komatsu H, Lauer G, Pybus OG, Ouchi K, Wong D, Ward S, Walker B, Klenerman P. 2006. Do antiviral CD8+ T cells select hepatitis C virus escape mutants? Analysis in diverse epitopes targeted by human intrahepatic CD8+ T lymphocytes. J Viral Hepat, 13 (2), pp. 121-130. | Show Abstract | Read more

Hepatitis C virus (HCV) is a variable RNA virus that can readily establish persistent infection. Cellular immune responses are important in the early control of the virus. Evidence from animal models suggests that mutation in epitopes recognized by CD8+ T lymphocytes may play an important role in the establishment of persistence but in human persistent infection, equivalent evidence is lacking. We investigated this by analysing a unique resource: viruses from a set of chronically HCV-infected individuals in whom the CD8+ T-cell responses in liver had previously been accurately mapped. Virus was sequenced in seven individuals at 10 epitopes restricted by 10 human leucocyte antigen (HLA) molecules. Two main patterns emerged: in the majority of epitopes sequenced, no variation was seen. In three epitopes, mutations were identified which were compatible with immune escape as assessed using phylogenetic and/or functional studies. These data suggest that - even where specific intrahepatic T cells are detectable - many epitopes do not undergo mutation in chronic human infection. On the contrary, virus may escape from intrahepatic CD8+ T-cell responses in a 'patchy' manner in certain specific epitopes. Furthermore, longitudinal studies to identify the differences between 'selecting' and 'nonselecting' intrahepatic CD8+ T-cell responses are needed in HCV infection.

Komatsu H, Inui A, Sogo T, Fujisawa T, Nagasaka H, Nonoyama S, Sierro S, Northfield J, Lucas M, Vargas A, Klenerman P. 2006. Large scale analysis of pediatric antiviral CD8+ T cell populations reveals sustained, functional and mature responses. Immun Ageing, 3 pp. 11. | Show Abstract | Read more

BACKGROUND: Cellular immunity plays a crucial role in cytomegalovirus (CMV) infection and substantial populations of CMV-specific T cells accumulate throughout life. However, although CMV infection occurs during childhood, relatively little is know about the typical quantity and quality of T cell responses in pediatric populations. METHODS: One thousand and thirty-six people (Male/Female = 594/442, Age: 0-19 yr.; 959 subjects, 20-29 yr.; 77 subjects) were examined for HLA typing. All of 1036 subjects were tested for HLA-A2 antigen. Of 1036 subjects, 887 were also tested for HLA-A23, 24 antigens. In addition, 50 elderly people (Male/Female = 11/39, Age: 60-92 yr.) were also tested for HLA-A2 antigen. We analyzed the CD8+ T cell responses to CMV, comparing these to responses in children and young. The frequencies, phenotype and function CD8+ T cells for two imunodominant epitopes from pp65 were measured. RESULTS: We observed consistently high frequency and phenotypically "mature" (CD27 low, CD28 low, CD45RA+) CMV-specific CD8+ T cell responses in children, including those studied in the first year of life. These CD8+ T cells retained functionality across all age groups, and showed evidence of memory "inflation" only in later adult life. CONCLUSION: CMV consistently elicits a very strong CD8+ T cell response in infants and large pools of CMV specific CD8+ T cells are maintained throughout childhood. The presence of CMV may considerably mould the CD8+ T cell compartment over time, but the relative frequencies of CMV-specific cells do not show the evidence of a population-level increase during childhood and adulthood. This contrast with the marked expansion ("inflation") of such CD8+ T cells in older adults. This study indicates that large scale analysis of peptide specific T cell responses in infants is readily possible. The robust nature of the responses observed suggests vaccine strategies aimed at priming and boosting CD8+ T cells against major pathogens (including HIV, malaria and CMV itself) could be successful in this age-group.

Isa A, Norbeck O, Hirbod T, Lundqvist A, Kasprowicz V, Bowness P, Klenerman P, Broliden K, Tolfvenstam T. 2006. Aberrant cellular immune responses in humans infected persistently with parvovirus B19. J Med Virol, 78 (1), pp. 129-133. | Show Abstract | Read more

A subset of parvovirus B19 (B19) infected patients retains the infection for years, as defined by detection of B19 DNA in bone marrow. Thus far, analysis of B19-specific humoral immune responses and viral genome variations has not revealed a mechanism for the absent viral clearance. In this study, ex-vivo cellular immune responses were assessed by enzyme linked immunospot assay mounted against the majority of the translated viral genome. Compared to seropositive healthy individuals, individuals with B19 persistence (2-8 years) showed larger number of responses to the structural proteins (P = 0.0022), whereas responses to the non-structural protein were of lower magnitude (P = 0.012). These observations provide the first findings of immunological discrepancies between individuals with B19 persistence and healthy individuals, findings that may reflect both failed immunity and antigenic exhaustion.

Isa A, Kasprowicz V, Norbeck O, Loughry A, Jeffery K, Broliden K, Klenerman P, Tolfvenstam T, Bowness P. 2005. Prolonged activation of virus-specific CD8+T cells after acute B19 infection. PLoS Med, 2 (12), pp. e343. | Show Abstract | Read more

BACKGROUND: Human parvovirus B19 (B19) is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. METHODS AND FINDINGS: The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA-peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%-0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. CONCLUSION: This is the first example to our knowledge of an "acute" human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation--analogous to that for cytomegalovirus infection--is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development.

Kasprowicz VO, Isa A, Norbeck O, Tolfvenstam T, Klenerman P, Bowness P. 2005. Striking TCR BV5.1 usage in parvovirus B19 specific HLA-A24 restricted FYTPLADQF-specific CD8+ T cells IMMUNOLOGY, 116 pp. 86-86.

Scriba TJ, Purbhoo M, Day CL, Robinson N, Fidler S, Fox J, Weber JN, Klenerman P, Sewell AK, Phillips RE. 2005. Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining. J Immunol, 175 (10), pp. 6334-6343. | Show Abstract

HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.

Baars PA, Sierro S, Arens R, Tesselaar K, Hooibrink B, Klenerman P, van Lier RA. 2005. Properties of murine (CD8+)CD27- T cells. Eur J Immunol, 35 (11), pp. 3131-3141. | Show Abstract | Read more

In humans, loss of CD27 expression is associated with the stable acquisition of effector functions by CD8+ T cells. We found that murine (CD8+)CD27- T cells were confined to the primed CD62L(dull/-)CD44(bright)CCR7- T cell population. (CD8+)CD27- T cells were absent from lymph nodes but could be found in blood, spleen and in non-lymphoid organs such as lung and liver. Late after primary influenza virus infection, low percentages of antigen-specific CD27- cells emerged in the lung and spleen. After recovery from secondary influenza virus infection, high percentages of influenza-specific CD27- T cells were found in the lung and the loss of CD27 on lung CD8+ T cells coincided with high granzyme B expression. After murine cytomegalovirus infection, loss of CD27 expression on virus-specific CD8+ T cell populations was sustained and especially marked in liver and lung. We suggest that in mice, CD27 is lost from CD8+ T cells only after repetitive antigenic stimulation. Moreover, the high expression of both granzyme B and perforin in the CD27- T cells suggests that the lack of CD27 on murine CD8+ T cells can be used to identify memory T cells with expression of cytotoxic effector molecules.

Meier UC, Owen RE, Taylor E, Worth A, Naoumov N, Willberg C, Tang K, Newton P et al. 2005. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections. J Virol, 79 (19), pp. 12365-12374. | Show Abstract | Read more

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.

Lauer GM, Lucas M, Timm J, Ouchi K, Kim AY, Day CL, Schulze Zur Wiesch J, Paranhos-Baccala G et al. 2005. Full-breadth analysis of CD8+ T-cell responses in acute hepatitis C virus infection and early therapy. J Virol, 79 (20), pp. 12979-12988. | Show Abstract | Read more

Multispecific CD8(+) T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8(+) T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8(+) T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8(+) T-cell responses, as well as CD4(+) T-cell responses. Rapid recrudescence also occurred despite broad CD8(+) T-cell responses. Importantly, in vivo suppression of CD3(+) T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8(+) T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.

Pillay T, Zhang HT, Drijfhout JW, Robinson N, Brown H, Khan M, Moodley J, Adhikari M et al. 2005. Unique acquisition of cytotoxic T-lymphocyte escape mutants in infant human immunodeficiency virus type 1 infection. J Virol, 79 (18), pp. 12100-12105. | Show Abstract | Read more

The role of cytotoxic T-lymphocyte (CTL) escape in rapidly progressive infant human immunodeficiency virus type 1 (HIV-1) infection is undefined. The data presented here demonstrate that infant HIV-1-specific CTL can select for viral escape variants very early in life. These variants, furthermore, may be selected specifically in the infant, despite the same CTL specificity being present in the mother. Additionally, pediatric CTL activity may be compromised both by the transmission of maternal escape variants and by mother-to-child transmission of escape variants that originally arose in the father. The unique acquisition of these CTL escape forms may help to explain the severe nature of some pediatric HIV infections.

Klenerman P, Hill A. 2005. T cells and viral persistence: lessons from diverse infections. Nat Immunol, 6 (9), pp. 873-879. | Show Abstract | Read more

Persistent virus infections create specific problems for their hosts. Although the dynamics of immune responses after acute infection are well studied and very consistent, especially in mouse models, the patterns of responses noted during persistent infection are more complex and differ depending on the infection. In particular, CD8(+) T cell responses differ widely in quantity and quality. In this review we examine these diverse responses and ask how they may arise; in particular, we discuss the function of antigen re-encounter and the CD4(+) T cell responses to and the escape strategies of specific viruses. We focus on studies of four main human pathogens, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and hepatitis C virus, and their animal models.

Norbeck O, Isa A, Pöhlmann C, Broliden K, Kasprowicz V, Bowness P, Klenerman P, Tolfvenstam T. 2005. Sustained CD8+ T-cell responses induced after acute parvovirus B19 infection in humans. J Virol, 79 (18), pp. 12117-12121. | Show Abstract | Read more

Murine models have suggested that CD8+ T-cell responses peak early in acute viral infections and are not sustained, but no evidence for humans has been available. To address this, we longitudinally analyzed the CD8+ T-cell response to human parvovirus B19 in acutely infected individuals. We observed striking CD8+ T-cell responses, which were sustained or even increased over many months after the resolution of acute disease, indicating that CD8+ T cells may play a prominent role in the control of parvovirus B19 and other acute viral infections of humans, including potentially those generated by live vaccines.

Jansson A, Barnes E, Klenerman P, Harlén M, Sørensen P, Davis SJ, Nilsson P. 2005. A theoretical framework for quantitative analysis of the molecular basis of costimulation. J Immunol, 175 (3), pp. 1575-1585. | Show Abstract

We present a theoretical framework for simulating the synaptic accumulation of the costimulatory molecules CD28, CTLA-4, B7-1, and B7-2, based on a system of mean-field, ordinary differential equations, and rigorous biophysical and expression data. The simulations show that binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects all profoundly influence complex formation at cellular interfaces. B7-2 engages 33-fold more CD28 than CTLA-4 at the synapse in contrast to B7-1, which ligates approximately 7-fold more CTLA-4 than CD28. Although B7-1 completely dominates interactions with CTLA-4, forming linear arrays of 7-18 receptor-ligand pairs, CTLA-4 is fully engaged by B7-2 when B7-1 is absent. Additional simulations reveal the sensitivity of CD28 interactions to modeled transport processes. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be due to the differential binding properties of B7-1 and B7-2 only. We discuss the apparent redundancy of B7-1 in the context of a potentially dynamic synaptic microenvironment, and in light of functions other than the direct enhancement of T cell inhibition by CTLA-4.

Harcourt GC, Donfield S, Gomperts E, Daar ES, Goulder P, Phillips RE, Klenerman P, Hemophilia Growth and Development Study (HGDS). 2005. Longitudinal analysis of CD8 T-cell responses to HIV and hepatitis C virus in a cohort of co-infected haemophiliacs. AIDS, 19 (11), pp. 1135-1143. | Show Abstract | Read more

OBJECTIVE: To investigate CD8 T-cell responses to HIV and hepatitis C virus (HCV) over time in a group of co-infected children with haemophilia to assess the influence of the virus infections on each other and on clinical outcome. DESIGN: The HIV and HCV CD8 T-cell response of HLA-A2 co-infected individuals in the cohort were analysed at two time points, looking at the frequency and phenotype of HIV-specific T cells and assessing overall responses to the two viruses. METHODS: Peripheral blood mononuclear cells (PBMC) from 72 HLA-A2 co-infected individuals were analysed using an HIV HLA-A2 tetramer and by IFN-gamma ELISpot using a panel of HIV and HCV antigens. PBMC from a group of 26 HLA-A2 HIV mono-infected adults were also analysed as a comparison. RESULTS: We identified two distinct patterns of response: some patients had a limited response to either virus whilst others made responses to a range of HIV epitopes. HCV responses were detected only in those who made multiple responses to HIV epitopes (P<0.0001). HCV infection had an influence on the phenotype of HIV-specific CD8 T cells, with a reduction in relative perforin and CD57 expression. Lack of functional or tetramer-positive HIV-specific T cells was associated with a decline in absolute CD4 T-cell counts between the time points (up to 7 years; P = 0.005). CONCLUSION: HCV infection has an impact on the phenotype of HIV-specific CD8 T cells. In this well-defined cohort, failure to maintain effective CD8 T-cell responses against HIV may contribute to disease progression.

Feeney ME, Tang Y, Pfafferott K, Roosevelt KA, Draenert R, Trocha A, Yu XG, Verrill C et al. 2005. HIV-1 viral escape in infancy followed by emergence of a variant-specific CTL response. J Immunol, 174 (12), pp. 7524-7530. | Show Abstract

Mutational escape from the CTL response represents a major driving force for viral diversification in HIV-1-infected adults, but escape during infancy has not been described previously. We studied the immune response of perinatally infected children to an epitope (B57-TW10) that is targeted early during acute HIV-1 infection in adults expressing HLA-B57 and rapidly mutates under this selection pressure. Viral sequencing revealed the universal presence of escape mutations within TW10 among B57- and B5801-positive children. Mutations in TW10 and other B57-restricted epitopes arose early following perinatal infection of B57-positive children born to B57-negative mothers. Surprisingly, the majority of B57/5801-positive children exhibited a robust response to the TW10 escape variant while recognizing the wild-type epitope weakly or not at all. These data demonstrate that children, even during the first years of life, are able to mount functional immune responses of sufficient potency to drive immune escape. Moreover, our data suggest that the consequences of immune escape may differ during infancy because most children mount a strong variant-specific immune response following escape, which is rarely seen in adults. Taken together, these findings indicate that the developing immune system of children may exhibit greater plasticity in responding to a continually evolving chronic viral infection.

Rushbrook SM, Ward SM, Unitt E, Vowler SL, Lucas M, Klenerman P, Alexander GJ. 2005. Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection. J Virol, 79 (12), pp. 7852-7859. | Show Abstract | Read more

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.

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Rushbrook SM, Ward SM, Unitt E, Vowler SL, Lucas M, Klenerman P, Alexander GJM. 2005. Regulatory T cells suppress in vitro proliferation of virus-specific CD8(+) T cells during persistent hepatitis C virus infection JOURNAL OF VIROLOGY, 79 (12), pp. 7852-7859. | Read more

Semmo N, Day CL, Ward SM, Lucas M, Harcourt G, Loughry A, Klenerman P. 2005. Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection. Hepatology, 41 (5), pp. 1019-1028. | Show Abstract | Read more

Hepatitis C virus (HCV) becomes persistent in the majority of infected individuals. In doing so, the virus evades host adaptive immune responses, although the mechanisms responsible in this evasion are not clear. Several groups have demonstrated weak or absent HCV-specific CD4+ T cell responses during chronic HCV infection using proliferation assays and, more recently, class II tetramers. However, the functional status of HCV-specific CD4+ T cells in resolved and persistent infection is poorly understood. Using interferon gamma (IFN-gamma) and interleukin 2 (IL-2) enzyme-linked immunospot assays, we analyzed cytokine secretion patterns in chronically infected patients and compared them with those with resolved infection. In the spontaneous resolver group, strong IL-2 secretion in relation to IFN-gamma secretion was observed. However, in the persistently infected group, a consistent and significant loss of IL-2-secreting cells, compared with IFN-gamma-secreting cells, was identified. In vitro addition of IL-2 had a substantial effect in restoring CD4+ T cell activity. In conclusion, failure of IL-2 secretion, as opposed to physical deletion or complete functional unresponsiveness, appears to be an important determinant of the status of CD4+ T cell populations in chronic HCV infection. Loss of IL-2 secretory capacity may lead to disruption of IFN-gamma and proliferative function in vivo-a status that characterizes the cellular immune response in both CD4+ and CD8+ compartments in chronic disease.

Semmo N, Harcourt G, Klenerman P, Taylor C, Smith N. 2005. HCV-indeterminate blood donors or occult HCV infection? Reply LANCET, 365 (9470), pp. 1541-1541. | Read more

Northfield J, Lucas M, Jones H, Young NT, Klenerman P. 2005. Does memory improve with age? CD85j (ILT-2/LIR-1) expression on CD8 T cells correlates with 'memory inflation' in human cytomegalovirus infection. Immunol Cell Biol, 83 (2), pp. 182-188. | Show Abstract | Read more

CMV-specific memory CD8(+) T cells accumulate over time to reach high frequencies amongst peripheral blood lymphocytes - a phenomenon termed 'memory inflation'. Using tetramer staining on samples from a large number of subjects and multivariate regression analysis, we were able to relate this to the phenotype of CD8(+) T cells. We made the following observations: (i) CD85j (ILT-2/LIR-1) was highly expressed alongside CD57 - an established effector memory marker - on CMV-specific CD8(+) T cells; (ii) on CD8(+) T cells as a whole, with increasing age, CD57 and CD85j (ILT-2/LIR-1) expression increased whereas CCR7 expression decreased, indicating increasing maturation of the total CD8(+) T-cell compartment with age; (iii) unit increases in the percentage of CMV-specific CD8(+) T cells expressing CD57 and CD85j (ILT-2/LIR-1) were associated with incremental expansion of these T-cell populations; (iv) CMV seropositivity is associated with a marked effect on the overall phenotype of CD8(+) T cells (at any given age, CMV seropositivity is associated with an 18.7% increase in CD85j (ILT-2/LIR-1) expression); and (v) from our observations we estimated from this an apparent 'ageing effect' of CMV on CD8(+) T cells of 35.4 years. The data presented are consistent with a predictable, unidirectional and linear model of virus specific T-cell differentiation and maturation.

Sierro S, Rothkopf R, Klenerman P. 2005. Evolution of diverse antiviral CD8+ T cell populations after murine cytomegalovirus infection. Eur J Immunol, 35 (4), pp. 1113-1123. | Show Abstract | Read more

Cytomegalovirus (CMV) is a major human pathogen normally controlled by cellular immune responses. The infection can be modeled in the mouse using murine CMV (MCMV). During the latent phase of infection, two different patterns of CD8(+) T cell responses have been observed: some specificities show increasing frequencies over time ("memory inflation"), while others, which are present acutely, are barely detectable at later time points. This distinction is independent of initial immunodominance. We analyzed the extent to which such responses differ functionally and tracked both their population distribution and their evolution over time. We observed two clear patterns of memory development that diverged early after infection. Acutely, CD8(+) T cells directed against all epitopes showed similar activation, phenotype and distribution. Thereafter, one set of responses ("inflationary") increased in frequency over time, was found in high numbers in non-lymphoid organs and was associated with an activated (CD28(low) CD27(low)CD122(low)) phenotype. In contrast, CD8(+) T cells responses specific for other MCMV epitopes ("non-inflationary") showed a slow reversion to a classical "central" memory phenotype without enrichment in non-lymphoid organs. A simple model to describe the equilibrium state in MCMV is presented, which may point to previously unexplored antiviral populations present after human CMV infection.

Clayton RF, Rinaldi A, Kandyba EE, Edward M, Willberg C, Klenerman P, Patel AH. 2005. Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int, 25 (2), pp. 389-402. | Show Abstract | Read more

BACKGROUND: Liver cell lines closely resembling primary hepatocyte are essential for research on hepatitis viruses and hepatocyte function. Currently used cell lines are derived from hepatic tumours and have altered gene expression. AIMS: The generation and characterisation of novel human hepatocyte lines (HHLs) derived from healthy human liver, retaining the primary hepatocyte phenotype. RESULTS: Primary hepatocytes were immortalised with Moloney's mouse leukaemia virus expressing E6 and E7 proteins of human papillomavirus, and cultures propagated long-term. All HHLs contained markers of hepatocyte and biliary phenotype (cytokeratins 7, 8, 18 and 19), Cytochrome P450 and albumin. The HHLs did not express high levels of p53 or alpha-fetoprotein. When grown in a collagen sandwich culture, or at the air-liquid interface, HHLs were maintained as monolayer whereas Huh-7 and HepG2 formed thick layers. All HHLs showed increased capacity to bind recombinant hepatitis C virus-like particles in comparison with Huh-7 and HepG2. We also demonstrate that HHLs contained active gap junctions, and that the cells respond to stimulation with IFN-alpha by upregulation of major histocompatibility complex (MHC)-I and -II. CONCLUSIONS: These HHLs retain primary hepatocyte phenotype and should be useful for investigating mechanisms of entry and replication of hepatotropic viruses, and should also be valuable in the study of hepatocyte biology and pathology.

Leslie A, Kavanagh D, Honeyborne I, Pfafferott K, Edwards C, Pillay T, Hilton L, Thobakgale C et al. 2005. Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA. J Exp Med, 201 (6), pp. 891-902. | Show Abstract | Read more

Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of "negatively associated" or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

Kim AY, Lauer GM, Ouchi K, Addo MM, Lucas M, Schulze Zur Wiesch J, Timm J, Boczanowski M et al. 2005. The magnitude and breadth of hepatitis C virus-specific CD8+ T cells depend on absolute CD4+ T-cell count in individuals coinfected with HIV-1. Blood, 105 (3), pp. 1170-1178. | Show Abstract | Read more

CD8(+) T-cell responses are an essential antiviral host defense in persistent viral infections, and their sustained effectiveness is thought to be critically dependent on CD4(+) T-helper cells. To determine the relationship between HIV-1-induced CD4(+) T-cell depletion and hepatitis C virus (HCV)-specific CD8(+) T-cell responses during viral persistence, we studied 103 persons positive for HCV, 74 coinfected with HIV-1. CD8(+) T-cell responses to the entire HCV polyprotein were determined by using an interferon-gamma enzyme-linked immunospot (ELISpot) assay. Although HIV-1 infection by itself was not associated with a diminished HCV-specific response, HIV-1-associated CD4(+) depletion was associated with significantly lower HCV-specific CD8(+) T cells (R = 0.48, P < .0001). In contrast, declining CD4(+) counts over the same range were not associated with diminished Epstein-Barr virus (EBV)- (R = 0.19, P = .31) or HIV-1-specific (R = -0.13, P = .60) CD8(+) T-cell responses in persons infected with all viruses. These data indicate that frequencies of circulating HCV-specific CD8(+) T-cell responses are sensitive to absolute CD4(+) T-cell counts and provide a possible explanation for the accelerated HCV disease course in persons coinfected with HIV-1 and HCV.

Scriba TJ, Zhang HT, Brown HL, Oxenius A, Tamm N, Fidler S, Fox J, Weber JN et al. 2005. HIV-1-specific CD4+ T lymphocyte turnover and activation increase upon viral rebound. J Clin Invest, 115 (2), pp. 443-450. | Show Abstract | Read more

HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1-specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.

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Scriba TJ, Zhang HT, Brown HL, Oxenius A, Tamm N, Fidler S, Fox J, Weber JN et al. 2005. HIV-1-specific CD4(+) T lymphocyte turnover and activation increase upon viral rebound JOURNAL OF CLINICAL INVESTIGATION, 115 (2), pp. 443-450. | Show Abstract | Read more

HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1-specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.

Semmo N, Barnes E, Taylor C, Kurtz J, Harcourt G, Smith N, Klenerman P. 2005. T-cell responses and previous exposure to hepatitis C virus in indeterminate blood donors. Lancet, 365 (9456), pp. 327-329. | Show Abstract | Read more

Blood donors are routinely screened for hepatitis C virus infection. Some individuals have weak or restricted virus-specific antibody responses, and are classed as indeterminate. Such donors are almost always negative for viral RNA in blood. We postulated that previous transient virus exposure might account for some of these cases. With sensitive ex-vivo analyses of T-cell responses, we identified virus-specific responses in 15 of 30 indeterminate blood donors tested, compared with none in controls (p=0.0013). Additionally, these responses were typically focused on core-derived peptides. These findings suggest previous exposure to the virus in many indeterminate blood donors.

Northfield JW, Harcourt G, Lucas M, Klenerman P. 2005. Immunology of viral co-infections with HIV. Arch Immunol Ther Exp (Warsz), 53 (1), pp. 3-12. | Show Abstract

Increasing clinical evidence is emerging that other persistent viral infections can act as important co-factors affecting the progression of human munodeficiency virus-1 (HIV-1). It appears that hepatitis C (HCV) and cytomegalovirus (CMV) have a deleterious effect on HIV progression, whereas hepatitis G (GBV-C) benefits HIV-1 progression. At the same time, the aggressive nature of HCV infection in HIV is clearly recognized. Here we discuss this clinical evidence and go on to review scientific work pertaining to these interactions in the context of the known and theoretical immunological effects of these viruses. This is discussed at the level of the generation of adaptive immune responses and their effector functions. It is clear that co-infection with persistent viral infections may pose special problems for the human immune system, as pathogenic effects may not be specific to the actual eliciting virus and can therefore multiply the difficulties faced by host defenses. We also highlight the need for further therapies for HIV/HCV co-infected persons, as this is currently a complex and severe syndrome.

Quiroga JA, Carreño V. 2005. HCV-indeterminate blood donors or occult HCV infection? Lancet, 365 (9470), pp. 1540-1541. | Read more

Timm J, Lauer GM, Kavanagh DG, Sheridan I, Kim AY, Lucas M, Pillay T, Ouchi K et al. 2004. CD8 epitope escape and reversion in acute HCV infection. J Exp Med, 200 (12), pp. 1593-1604. | Show Abstract | Read more

In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, Chetty S, Rathnavalu P, Moore C et al. 2004. Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature, 432 (7018), pp. 769-775. | Show Abstract | Read more

The extreme polymorphism in the human leukocyte antigen (HLA) class I region of the human genome is suggested to provide an advantage in pathogen defence mediated by CD8+ T cells. HLA class I molecules present pathogen-derived peptides on the surface of infected cells for recognition by CD8+ T cells. However, the relative contributions of HLA-A and -B alleles have not been evaluated. We performed a comprehensive analysis of the class I restricted CD8+ T-cell responses against human immunodeficiency virus (HIV-1), immune control of which is dependent upon virus-specific CD8+ T-cell activity. In 375 HIV-1-infected study subjects from southern Africa, a significantly greater number of CD8+ T-cell responses are HLA-B-restricted, compared to HLA-A (2.5-fold; P = 0.0033). Here we show that variation in viral set-point, in absolute CD4 count and, by inference, in rate of disease progression in the cohort, is strongly associated with particular HLA-B but not HLA-A allele expression (P < 0.0001 and P = 0.91, respectively). Moreover, substantially greater selection pressure is imposed on HIV-1 by HLA-B alleles than by HLA-A (4.4-fold, P = 0.0003). These data indicate that the principal focus of HIV-specific activity is at the HLA-B locus. Furthermore, HLA-B gene frequencies in the population are those likely to be most influenced by HIV disease, consistent with the observation that B alleles evolve more rapidly than A alleles. The dominant involvement of HLA-B in influencing HIV disease outcome is of specific relevance to the direction of HIV research and to vaccine design.

Lauer GM, Barnes E, Lucas M, Timm J, Ouchi K, Kim AY, Day CL, Robbins GK et al. 2004. High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection. Gastroenterology, 127 (3), pp. 924-936. | Show Abstract | Read more

BACKGROUND & AIMS: Cellular immune responses are thought to play a key role in the resolution of primary HCV infection. Although it has been consistently shown that CD4+ T-cell responses are maintained in those with spontaneous resolution but lost in those with persistent infection, the role of CD8+ T-cell responses remains controversial. Previous studies have largely focused on limited HLA alleles and predefined CD8+ T-cell epitopes, and, thus, comprehensive studies remain to be performed. METHODS: To understand the composition of the immune response associated with spontaneous resolution, we comprehensively mapped CD8+ T-cell responses in 20 HLA-diverse persons with resolved HCV infection, using HCV peptides spanning the entire genome. We analyzed the magnitude, breadth, function, and phenotype using ELISpot, class-I tetramers, intracellular cytokine staining, and cytolytic assays. We studied in parallel HCV-specific responses and viral sequence variation in persistent infection. RESULTS: Responses in individuals with resolved infection were strong and broad with robust proliferation in response to antigen. Responses in those persistently infected were rarely detected ex vivo and, when present, were narrowly directed and weak. However, they also proliferated in vitro. Dominant target epitopes differed among individuals in both cohorts, despite frequently shared HLA-alleles. CONCLUSIONS: These data indicate that persisting, strong CD8+ T-cell responses are observed in the majority of persons with resolved HCV infection and provide support for strategies to boost CD8+ T-cell responses for the prevention or treatment of HCV infection but also highlight the diversity of responses that may need to be elicited to provide protection.

Barnes E, Salio M, Cerundolo V, Medlin J, Murphy S, Dusheiko G, Klenerman P. 2004. Impact of alpha interferon and ribavirin on the function of maturing dendritic cells. Antimicrob Agents Chemother, 48 (9), pp. 3382-3389. | Show Abstract | Read more

Alpha interferon and ribavirin are required in combination to achieve a sustained virological response in the treatment of hepatitis C virus (HCV) infection. Alpha interferon has direct antiviral activity and also enhances HCV-specific T-cell responses. Ribavirin has little direct activity against HCV but reduces hepatic inflammation. It is therefore likely that these drugs in combination have hitherto unidentified immunological effects. In the present study we investigated the effects of alpha interferon and ribavirin on dendritic cell (DC) maturation and cytokine production induced by double-stranded RNA in vitro. Alpha interferon alone enhanced the expression of HLA class I, HLA class II, and CD86 on immature DCs but did not stimulate full DC maturation, which requires the expression of CD83. Alpha interferon enhanced the production of interleukin 12 p70 [IL-12(p70)] and tumor necrosis factor alpha (TNF-alpha) but had no effect on IL-10 production. In contrast, ribavirin at physiological doses had no effect on DC maturation but markedly suppressed the production of TNF-alpha, IL-10, and IL-12(p70). The suppression of cytokines by ribavirin cannot be explained by the induction of DC apoptosis or cell death. Quantitative PCR confirmed that cytokine suppression occurs at the level of mRNA. The suppression of IL-12(p70) and TNF-alpha in maturing DCs may explain the reduction in hepatic inflammation observed during ribavirin monotherapy. Combination alpha interferon-ribavirin therapy may alter the cytokine profile of maturing DCs overall by suppressing IL-10 production but maintaining IL-12(p70) and TNF-alpha production, a pattern that would favor viral elimination through downstream effects on T cells.

Harcourt GC, Lucas M, Sheridan I, Barnes E, Phillips R, Klenerman P. 2004. Longitudinal mapping of protective CD4+ T cell responses against HCV: analysis of fluctuating dominant and subdominant HLA-DR11 restricted epitopes. J Viral Hepat, 11 (4), pp. 324-331. | Show Abstract | Read more

Cellular immunity plays an important role in the control of persistent virus infections such as hepatitis C virus (HCV). Antiviral CD4(+) T cell responses have been shown to accompany resolution of acute disease and there is also a consistent association between HLA Class II genes, notably HLADRB1*1101 (and the closely linked HLADQB1*0301) and disease resolution. We initially mapped longitudinal CD4(+) T cell responses in an individual after spontaneous resolution of acute HCV, and identified three HLA-DR11-restricted responses which vary in immunodominance over time. Functional assays and HLA Class II tetramer staining revealed one to be a response to a commonly recognized epitope, NS3(1248-1261), although cytokine capture assays showed these specific cells to be at a very low frequency. In this patient, and in others reported, this most frequently recognized HLA-DR11 restricted epitope is not immunodominant. We analysed whether sequence variability within and between genotypes might account for differences in recognition of HLA-DR11 restricted epitopes. We found that a limited number, including NS3(1248-1261), showed extreme sequence conservation. Within NS3, the ability of peptides to accept amino acid substitutions was clearly related to the structure of the protein. Overall the data provide a deeper understanding of the relationship between protein structure and variability of HLA-DR11 restricted peptides and may explain the apparent dominance of responses to NS3(1248-1261) across studies but not within an individual immune response.

Lucas M, Day CL, Wyer JR, Cunliffe SL, Loughry A, McMichael AJ, Klenerman P. 2004. Ex vivo phenotype and frequency of influenza virus-specific CD4 memory T cells. J Virol, 78 (13), pp. 7284-7287. | Show Abstract | Read more

Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct "central memory" CD62L(+) phenotype.

Bocharov G, Ludewig B, Bertoletti A, Klenerman P, Junt T, Krebs P, Luzyanina T, Fraser C, Anderson RM. 2004. Underwhelming the immune response: Effect of slow virus growth on CD(8+) T-lymphocyte responses JOURNAL OF VIROLOGY, 78 (11), pp. 6079-6079. | Read more

Barnes E, Ward SM, Kasprowicz VO, Dusheiko G, Klenerman P, Lucas M. 2004. Ultra-sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells. Eur J Immunol, 34 (6), pp. 1570-1577. | Show Abstract | Read more

A major breakthrough in cellular immunology has been the development of HLA class I tetramers to analyze CD8(+) T cell responses. However, in many situations, including persistent virus infection, specific T cell responses are rarely detected using this technology. This raises the question of whether such responses are 'deleted' (or 'exhausted') or present below the conventional detection limit for class I tetramer staining. In particular, persistent hepatitis C virus (HCV) infection is characterized by very weak or apparently absent specific CD8(+) T cell responses, even though they are readily detectable in acute disease. Therefore, we assessed the use of anti-PE-labeled magnetic beads to enrich tetramer-positive HCV-specific T cells and identify previously undetectable populations. Using the enrichment technique, HCV-specific T cells could be detected in the majority of infected individuals, whereas these responses were not detected using conventional tetramer staining (8/15 vs. 1/15; p=0.01). Magnetic enrichment could reliably detect very rare HCV-specific responses at frequencies of >0.0011% of CD8(+) T cells (approximately 1/million PBMC), and phenotypic analysis of these rare populations was possible. Therefore, this direct ex vivo technique revealed the persistence of very low frequencies of virus-specific CD8(+) T cells during chronic virus infection and is readily transferable to the study of other viral, self- or tumor-specific T cells.

Ward SM, Jonsson JR, Sierro S, Clouston AD, Lucas M, Vargas AL, Powell EE, Klenerman P. 2004. Virus-specific CD8+ T lymphocytes within the normal human liver. Eur J Immunol, 34 (6), pp. 1526-1531. | Show Abstract | Read more

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (V alpha 24/V beta 11) was also observed, a finding which may be relevant to the regulation of the antiviral populations.

Sheridan I, Pybus OG, Holmes EC, Klenerman P. 2004. High-resolution phylogenetic analysis of hepatitis C virus adaptation and its relationship to disease progression. J Virol, 78 (7), pp. 3447-3454. | Show Abstract | Read more

Hepatitis C virus (HCV) persists in the majority of those infected despite host immune responses. Evidence has accrued that selectively fixed mutations in the envelope genes (E1 and E2) are associated with viral persistence, particularly those that occur within the first hypervariable region of E2 (HVR1). However, the individual amino acid residues under selection have not been identified, nor have their selection pressures been measured, despite the importance of this information for understanding disease pathogenesis and for vaccine design. We performed a high-resolution analysis of published gene sequence data from individuals undergoing acute HCV infection, employing two phylogenetic methods to determine site-specific selection pressures. Strikingly, we found a statistically significant association between the number of sites selected and disease outcome, with the fewest selected sites in fulminant HCV cases and the greatest number of selected sites in rapid progressors, reflecting the duration and intensity of the arms race between host and virus. Moreover, sites outside the HVR1 appear to play a major role in viral evolution and pathogenesis, although there was no association between viral persistence and specific mutations in E1 and E2. Our analysis therefore allows fine dissection of immune selection pressures, which may be more diverse than previously thought. Such analyses could play a similarly informative role in studies of other persistent virus infections, such as human immunodeficiency virus.

Karrer U, Wagner M, Sierro S, Oxenius A, Hengel H, Dumrese T, Freigang S, Koszinowski UH, Phillips RE, Klenerman P. 2004. Expansion of protective CD8+ T-cell responses driven by recombinant cytomegaloviruses. J Virol, 78 (5), pp. 2255-2264. | Show Abstract | Read more

CD8(+) T cells are critical for the control of many persistent viral infections, such as human immunodeficiency virus, hepatitis C virus, Epstein-Barr virus, and cytomegalovirus (CMV). In most infections, large CD8(+)-T-cell populations are induced early but then contract and are maintained thereafter at lower levels. In contrast, CD8(+) T cells specific for murine CMV (MCMV) have been shown to gradually accumulate after resolution of primary infection. This unique behavior is restricted to certain epitopes, including an immunodominant epitope derived from the immediate-early 1 (IE1) gene product. To explore the mechanism behind this further, we measured CD8(+)-T-cell-mediated immunity induced by recombinant MCMV-expressing epitopes derived from influenza A virus or lymphocytic choriomeningitis virus placed under the control of an IE promoter. We observed that virus-specific CD8(+)-T-cell populations were induced and that these expanded gradually over time. Importantly, these CD8(+) T cells provided long-term protection against challenge without boosting. These results demonstrate a unique pattern of accumulating T cells, which provide long-lasting immune protection, that is independent of the initial immunodominance of the epitope and indicates the potential of T-cell-inducing vaccines based on persistent vectors.

Bocharov G, Ludewig B, Bertoletti A, Klenerman P, Junt T, Krebs P, Luzyanina T, Fraser C, Anderson RM. 2004. Underwhelming the immune response: effect of slow virus growth on CD8+-T-lymphocyte responses. J Virol, 78 (5), pp. 2247-2254. | Show Abstract | Read more

The speed of virus replication has typically been seen as an advantage for a virus in overcoming the ability of the immune system to control its population growth. Under some circumstances, the converse may also be true: more slowly replicating viruses may evoke weaker cellular immune responses and therefore enhance their likelihood of persistence. Using the model of lymphocytic choriomeningitis virus (LCMV) infection in mice, we provide evidence that slowly replicating strains induce weaker cytotoxic-T-lymphocyte (CTL) responses than a more rapidly replicating strain. Conceptually, we show a "bell-shaped" relationship between the LCMV growth rate and the peak CTL response. Quantitative analysis of human hepatitis C virus infections suggests that a reduction in virus growth rate between patients during the incubation period is associated with a spectrum of disease outcomes, from fulminant hepatitis at the highest rate of viral replication through acute resolving to chronic persistence at the lowest rate. A mathematical model for virus-CTL population dynamics (analogous to predator [CTL]-prey [virus] interactions) is applied in the clinical data-driven analysis of acute hepatitis B virus infection. The speed of viral replication, through its stimulus of host CTL responses, represents an important factor influencing the pathogenesis and duration of virus persistence within the human host. Viruses with lower growth rates may persist in the host because they "sneak through" immune surveillance.

Zafiropoulos A, Barnes E, Piggott C, Klenerman P. 2004. Analysis of 'driver' and 'passenger' CD8+ T-cell responses against variable viruses. Proc Biol Sci, 271 Suppl 3 (Suppl_3), pp. S53-S56. | Show Abstract | Read more

Variable viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), persist despite host immune responses directed against them. Numerous lines of evidence have suggested that antiviral CD8+ T-cell responses are key among these immune responses, but these vary widely in their ability to contain virus. We propose that only a proportion of responses may exert significant antiviral pressure ('driver' responses), leading to control over viral replication (protection) and/or, ultimately, selection of escape mutants. Another set of responses may exert only weak pressure on the virus ('passenger' responses): these neither protect nor select. To examine this we have analysed (using established databases of HIV and HCV sequences and cytotoxic T-lymphocyte (CTL) epitopes, and published experimental datasets) two important features--predicted binding of the epitope to major histocompatibility complex molecule and observed variability of the epitope--that might distinguish such responses. We find that a high predicted binding estimate could only explain a limited set of 'driver' responses associated with protection or selection. There is statistical evidence that readily defined (and non-protective) CTL responses target regions associated with lower levels of viral variability. Taken together, this suggests that a large number of well-documented responses may represent 'passengers' and we propose a mechanism that might explain their presence.

Klenerman P. 2004. T cells get by with a little help from their friends EUROPEAN JOURNAL OF IMMUNOLOGY, 34 (2), pp. 313-316. | Show Abstract | Read more

In animal models, lymphocytic choriomeningitis virus (LCMV) may be controlled after acute infection or may establish various levels of persistence. Cytotoxic responses mediated by CD8+ T cells are responsible for both initial control of LCMV and for immunopathology. As discussed in this article, there is emerging evidence that the levels of antigen to which the immune system is exposed over time are important in controlling CD8+ T cell activation, memory responses and exhaustion, and that these levels are affected by the efficiency of T cell help and the presence of antibody. To enable lasting control of LCMV infection, CD8+ T cells, CD4+ T cell help and B cells are all required. These findings have important implications for the prevention and treatment of infection by viruses such as hepatitis B and C viruses, cytomegalovirus and HIV. © 2004 Wiley-VCH Verlag GmbH & Co. KGaA.

Klenerman P. 2004. Commentary: T cells get by with a little help from their friends. Eur J Immunol, 34 (2), pp. 313-316. | Show Abstract | Read more

In animal models, lymphocytic choriomeningitis virus (LCMV) may be controlled after acute infection or may establish various levels of persistence. Cytotoxic responses mediated by CD8(+) T cells are responsible for both initial control of LCMV and for immunopathology. As discussed in this article, there is emerging evidence that the levels of antigen to which the immune system is exposed over time are important in controlling CD8(+) T cell activation, memory responses and exhaustion, and that these levels are affected by the efficiency of T cell help and the presence of antibody. To enable lasting control of LCMV infection, CD8(+) T cells, CD4(+) T cell help and B cells are all required. These findings have important implications for the prevention and treatment of infection by viruses such as hepatitis B and C viruses, cytomegalovirus and HIV. See accompanying article http://dx.doi.org/10.1002/eji.200324717

Lucas M, Vargas-Cuero AL, Lauer GM, Barnes E, Willberg CB, Semmo N, Walker BD, Phillips R, Klenerman P. 2004. Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8+ T cells. J Immunol, 172 (3), pp. 1744-1753. | Show Abstract

Recent studies using MHC class I tetramers have shown that CD8(+) T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8(+) T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8(+) T cells between these groups. In the HCV-infected cohort, CMV-specific CD8(+) T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8(+) T cell population, a novel feature that may be a hallmark of this infection.

Torti C, Barnes E, Quiros-Roldan E, Puoti M, Carosi G, Klenerman P. 2004. Suppression of hepatitis C virus replication is maintained long term following HAART therapy, in an individual with HCV/HIV co-infection. Antivir Ther, 9 (1), pp. 139-142.

Bocharov G, Klenerman P, Ehl S. 2004. Modelling the dynamics of LCMV infection in mice: II. Compartmental structure and immunopathology (vol 221, pg 349, 2003) JOURNAL OF THEORETICAL BIOLOGY, 226 (1), pp. 123-123. | Read more

Bocharov G, Ludewig B, Bertoletti A, Klenerman P, Junt T, Krebs P, Luzyanina T, Fraser C, Anderson RM. 2004. Erratum: Underwhelming the immune response: Effect of slow virus growth on CD8+-T-lymphocyte responses (Journal of Virology (2004) 78, 5 (2247-2254)) Journal of Virology, 78 (11), pp. 6079. | Read more

Bocharov G, Klenerman P, Ehl S. 2004. Erratum to “Modelling the dynamics of LCMV infection in mice: II. Compartmental structure and immunopathology” Journal of Theoretical Biology, 226 (1), pp. 123-123. | Read more

Klenerman P, Semmo N, Ward S. 2004. Cellular immune responses against HCV: T cells take a diversion in the liver Hepatology, 40 (6), pp. 1459-1461. | Read more

Hellier S, Frodsham AJ, Hennig BJ, Klenerman P, Knapp S, Ramaley P, Satsangi J, Wright M et al. 2003. Association of genetic variants of the chemokine receptor CCR5 and its ligands, RANTES and MCP-2, with outcome of HCV infection. Hepatology, 38 (6), pp. 1468-1476. | Show Abstract | Read more

The effect of host genetic variation on the outcome of hepatitis C virus (HCV) infection and its treatment is poorly understood. The chemokine receptors CCR5, CCR2, and CCR3 and their ligands, RANTES, MCP-1, MCP-2, and MIP-1alpha, are involved in the immune responses and the selective recruitment of lymphocytes to the liver in HCV infection. We studied 20 polymorphisms within these genes and investigated their association with persistent carriage of HCV, severity of liver disease, hepatic inflammation, and response to treatment in a large European cohort. Significant associations were found between CCR5-delta32 and reduced portal inflammation (P =.011, odds ratio [OR]: 2.3, 95% confidence interval [CI]: 1.09-4.84) and milder fibrosis (P =.015, OR: 1.97, 95% CI: 1.13-3.42). A promoter polymorphism at position -403 in the RANTES gene was associated with less severe portal inflammation (P =.004). An amino acid change in MCP2, Q46K, was associated with severity of fibrosis (P =.018, OR: 2.29, 95% CI: 1.14-4.58). In conclusion, our study suggests a possible role of the polymorphisms CCR5-delta32, RANTES -403, and MCP-2 Q46K in the outcome of HCV infection.

Karrer U, Sierro S, Wagner M, Oxenius A, Hengel H, Koszinowski UH, Phillips RE, Klenerman P. 2003. Memory inflation: Continous accumulation of antiviral CD8(+) T cells over time. (vol 170, pg 2022, 2003) JOURNAL OF IMMUNOLOGY, 171 (7), pp. 3895-3895.

Komatsu H, Sierro S, V Cuero A, Klenerman P. 2003. Population analysis of antiviral T cell responses using MHC class I-peptide tetramers. Clin Exp Immunol, 134 (1), pp. 9-12. | Show Abstract | Read more

MHC class I-peptide tetrameric complexes ('tetramers') have revolutionized the study of antiviral CD8+ T cell responses. They allow accurate quantification of immune responses ex vivo independent of function, with high levels of sensitivity. They have revealed unexpectedly large frequencies of 'memory' T cell responses against viruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), and provided information about their phenotypic and functional variation. However, such studies have generally concentrated on limited numbers of individuals analysed in detail. To allow larger population-based studies, we devised a method for tetramer analysis using 50-100 microlitre blood volumes in a 96-well plate format. We adapted this method to study the effect of age on responses in a cohort of nearly 600 individuals to an immunodominant HLA-A2 restricted response to CMV pp65 (NLVPMVATV). We observed the phenomenon of steady 'memory inflation' with age, similar to recently observed longitudinal data from murine studies. These data show that tetramers can be used as population screening tools and could be used to study age-related, geographical or seasonal effects in a number of other viral infections.

Day CL, Seth NP, Lucas M, Appel H, Gauthier L, Lauer GM, Robbins GK, Szczepiorkowski ZM et al. 2003. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J Clin Invest, 112 (6), pp. 831-842. | Show Abstract | Read more

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Cited:

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Day CL, Seth NP, Lucas M, Appel H, Gauthier L, Lauer GM, Robbins GK, Szczepiorkowski ZM et al. 2003. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers JOURNAL OF CLINICAL INVESTIGATION, 112 (6), pp. 831-842. | Show Abstract | Read more

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vβ repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Lamprecht P, Vargas Cuero AL, Muller A, Csernok E, Voswinkel J, Maass M, Solbach W, Gross WL, Klenerman P. 2003. Alterations in the phenotype of CMV-specific and total CD8+ T-cell populations in Wegener's granulomatosis. Cell Immunol, 224 (1), pp. 1-7. | Show Abstract | Read more

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.

Jones E, Price DA, Dahm-Vicker M, Cerundolo V, Klenerman P, Gallimore A. 2003. The influence of macrophage inflammatory protein-1alpha on protective immunity mediated by antiviral cytotoxic T cells. Immunology, 109 (1), pp. 68-75. | Show Abstract | Read more

Macrophage inflammatory protein 1alpha (MIP-1alpha), a member of the CC-chemokine subfamily, is known to induce chemotaxis of a variety of cell types in vivo. Although the role of MIP-1alpha in inflammatory responses generated following primary infection of mice with many different pathogens has been characterized, the influence of this chemokine on the generation of antigen-specific T-cell responses in vivo is less well understood. This is important, as virus-specific CD8+ T lymphocytes (CTL) play a crucial role in defence against viral infections, both acutely and in the long term. In this study, we compared the ability of wild-type and MIP-1alpha-deficient (MIP-1alpha-/-) mice to mount CTL responses specific for the immunodominant epitope derived from influenza nucleoprotein (NP366-374). Influenza-specific CTL responses were compared with respect to frequency, cytotoxic activity and ability to clear subsequent infections with recombinant vaccinia viruses expressing the influenza NP. The results indicate that antiviral CTL generated in MIP-1alpha-/- mice are slightly impaired in their ability to protect against a subsequent infection. However, impaired in vivo CTL-mediated antiviral protection was found to be associated with reduced cytotoxicity rather than with a failure of the CTL to migrate to peripheral sites of infection.

Bocharov G, Klenerman P, Ehl S. 2003. Modelling the dynamics of LCMV infection in mice: II. Compartmental structure and immunopathology. J Theor Biol, 221 (3), pp. 349-378. | Show Abstract | Read more

In this study, we develop a mathematical model for analysis of the compartmental aspects and immunopathology of lymphocytic choriomeningitis virus (LCMV) infection in mice. We used sets of original and published data on systemic (extrasplenic) virus distribution to estimate the parameters of virus growth and elimination for spleen and other anatomical compartments, such as the liver, kidney, thymus and lung as well as transfer rates between blood and the above organs. A mathematical model quantitatively integrating the virus distribution kinetics in the host, the specific cytotoxic T lymphocyte (CTL) response in spleen and the re-circulation of effector CTL between spleen, blood and liver is advanced to describe the CTL-mediated immunopathology (hepatitis) in mice infected with LCMV. For intravenous and "peripheral" routes of infection we examine the severity of the liver disease, as a function of the virus dose and the host's immune status characterized by the numbers of precursor and/or cytolytic effector CTL. The model is used to predict the efficacy of protection against virus persistence and disease in a localized viral infection as a function of the composition of CTL population. The modelling analysis suggests quantitative demands to CTL memory for maximal protection against a wide range of doses of infection with a primarily peripheral site of virus replication without the risk of favoring immunopathology. It specifies objectives for CTL vaccination to ensure virus elimination with minimal immunopathology vs. vaccination for disease.

Chakraborty R, Rees G, Bourboulia D, Cross AM, Dixon JR, D'Agostino A, Musoke R, Boshoff C, Rowland-Jones SL, Klenerman P. 2003. Viral coinfections among African children infected with human immunodeficiency virus type 1. Clin Infect Dis, 36 (7), pp. 922-924. | Show Abstract | Read more

City-dwelling children from Kenya who were infected with human immunodeficiency virus type 1 (HIV-1) were tested for coinfection with cytomegalovirus (CMV), human T cell lymphotropic viruses 1 and 2, Kaposi sarcoma-associated herpesvirus (KSHV), or hepatitis B, C, and G viruses. All children were found to be coinfected with CMV, whereas 5% had hepatitis G virus coinfection and 15% had KSHV coinfection. A protective role for hepatitis G virus cannot be excluded but likely affects only a minority of HIV-1-infected African children.

Lucas M, Tsitoura E, Montoya M, Laliotou B, Aslanoglou E, Kouvatsis V, Entwisle C, Miller J et al. 2003. Characterization of secreted and intracellular forms of a truncated hepatitis C virus E2 protein expressed by a recombinant herpes simplex virus. J Gen Virol, 84 (Pt 3), pp. 545-554. | Show Abstract | Read more

A replication-defective herpes simplex virus type 1 (HSV-1) recombinant lacking the glycoprotein H (gH)-encoding gene and expressing a truncated form of the hepatitis C (HCV) E2 glycoprotein (E2-661) was constructed and characterized. We show here that cells infected with the HSV/HCV recombinant virus efficiently express the HCV E2-661 protein. Most importantly, cellular and secreted E2-661 protein were both readily detected by the E2-conformational mAb H53 and despite the high expression levels, only limited amounts of misfolded aggregates were detected in either the cellular or secreted fractions. Furthermore, cell-associated and secreted E2-661 protein bound to the major extracellular loop (MEL) of CD81 in a concentration-dependent manner and both were highly reactive with sera from HCV-infected patients. Finally, BALB/c mice immunized intraperitoneally with the recombinant HSV/HCV virus induced high levels of anti-E2 antibodies. Analysis of the induced immunoglobulin G (IgG) isotypes showed high levels of IgG2a while the levels of the IgG1 isotype were significantly lower, suggesting a Th1-type of response. We conclude that the HSV-1 recombinant virus represents a promising tool for production of non-aggregated, immunologically active forms of the E2-661 protein and might have potential applications in vaccine development.

Komarova NL, Barnes E, Klenerman P, Wodarz D. 2003. Boosting immunity by antiviral drug therapy: a simple relationship among timing, efficacy, and success. Proc Natl Acad Sci U S A, 100 (4), pp. 1855-1860. | Show Abstract | Read more

Drug therapies against persistent human infections such as hepatitis C virus, hepatitis B virus, and HIV fail to consistently eradicate the infection from the host. Hence, recent emphasis has shifted to the study of antiviral therapy aimed at boosting specific immune responses. It was argued that structured therapy interruptions were required to achieve this, because such regimes have shown promising results in early HIV infection. Using mathematical models, we show that, contrary to this notion, a single phase of drug therapy can result in the establishment of sustained immunity. We present a simple relationship between timing of therapy and efficacy of the drugs required for success. In the presence of strong viral suppression, we show that therapy should be stopped relatively early, and that a longer duration of treatment leads to failure. On the other hand, in the presence of weaker viral suppression, stopping treatment too early is detrimental, and therapy has to be continued beyond a time threshold. We discuss our modeling results primarily in the context of HCV therapy during chronic infection. Although the therapy regimes explored here also have implications for HIV, virus-mediated destruction of specific immune cells renders success unlikely during the chronic phase of the infection.

Karrer U, Sierro S, Wagner M, Oxenius A, Hengel H, Koszinowski UH, Phillips RE, Klenerman P. 2003. Memory inflation: continuous accumulation of antiviral CD8+ T cells over time. J Immunol, 170 (4), pp. 2022-2029. | Show Abstract

CD8+ T lymphocytes play an important role in the control of intracellular pathogens during both acute and persistent infections. This is particularly true in the case of persistent herpesviruses such as human CMV, which are typified by large virus-specific CD8+ T cell populations during viral latency. To understand the origin of these populations and the factors shaping them over time, we investigated the CD8+ T cell response after murine CMV (MCMV) infection. The kinetics of the acute response were characterized by rapid expansion of activated T cells, followed by a contraction phase. Thereafter, we observed a striking pattern, where MCMV-specific memory CD8+ T cells steadily accumulated over time, with 20% of all CD8+ T cells at 1 year specific for one MCMV epitope. Accumulation of MCMV-specific CD8+ T lymphocytes was seen in all organs tested and was associated with continuous activation of specific CD8+ T lymphocytes, primarily within lymph nodes. The pattern of accumulation was observed in only two of five epitopes tested, and was accompanied by a gradual restriction in usage of the variable region of the TCR beta-chain over time. This novel pattern of a virus-specific CD8+ T cell response suggests that continuous or repetitive exposure to Ag can slowly mold memory T cell populations over time. This may be relevant for understanding the evolution of the large human CMV-specific CD8+ T cell populations seen in humans.

Lucas M, Gadola S, Meier U, Young NT, Harcourt G, Karadimitris A, Coumi N, Brown D, Dusheiko G, Cerundolo V, Klenerman P. 2003. Frequency and phenotype of circulating Valpha24/Vbeta11 double-positive natural killer T cells during hepatitis C virus infection. J Virol, 77 (3), pp. 2251-2257. | Show Abstract | Read more

Natural killer T (NKT) cells are thought to be involved in innate responses against infection. We investigated one specific type of NKT cell, Valpha24/Vbeta11 double positive, in hepatitis C virus (HCV) infection. Lower frequencies of this population were detected in the blood of HCV PCR-positive patients than in controls. Unlike Valpha24/Vbeta11 NKT cells found in blood, those in the liver appeared to be recently activated.

Harcourt GC, Lucas M, Godkin AJ, Kantzanou M, Phillips RE, Klenerman P. 2003. Evidence for lack of cross-genotype protection of CD4+ T cell responses during chronic hepatitis C virus infection. Clin Exp Immunol, 131 (1), pp. 122-129. | Show Abstract | Read more

CD4+ T lymphocyte responses are thought to play a major role in control of the hepatitis C virus (HCV). Few, however, have been mapped down to the level of peptide and HLA restriction. Furthermore, the ability of such T cells to respond to viruses which differ in genotype has not been addressed in detail. In most cases of persistent infection with HCV, CD4 proliferative responses are weak or absent. From a large cohort of persistently infected patients, we identified an individual with unusually robust and persistent responses in the face of chronic infection. We firstly mapped two peptide epitopes to regions of the nonstructural protein NS4 (aa1686-1705 and aa 1746-1765). However, in contrast to the genotype 1a derived antigens used for mapping, the infecting virus was identified as genotype 3a. Strikingly, the patient's CD4 response to these epitopes were specific only for the genotype 1a sequence, and did not recognize genotype 3a synthetic peptides. Serologic assays indicated that prior exposure to HCV of genotype 1 had occurred. This patient therefore maintains strong CD4 proliferative responses which are genotype specific and not cross-reactive. The apparent 'misdirection' of these nonprotective responses has important implications for the role of natural and vaccine induced CD4 responses in the face of variable viruses.

Willberg C, Barnes E, Klenerman P. 2003. HCV immunology--death and the maiden T cell. Cell Death Differ, 10 Suppl 1 (S1), pp. S39-S47. | Show Abstract | Read more

Cellular immune responses play an important role in the control of hepatitis C virus (HCV), although in the majority of cases they ultimately fail. We examine the mechanisms by which virus-specific T cells may interact with a cell that is infected with HCV and how this interaction may explain the success and failure of the immune response. As an infected cell presenting foreign antigen, the hepatocyte will interact with a large number of lymphocytes, both by direct cell to cell contact and by indirect means through the secretion of cytokines and chemokines. These interactions may lead on the one hand to the death of infected hepatocytes or suppression of viral replication and on the other hand to the death of T lymphocytes or down regulation of their function. We suggest that activation of lymphocytes in lymphoid organs leads to generation of effector T cells (positive loop), while at the same time presentation of antigen in the liver either on hepatocytes or other specialised antigen presenting cells depresses these responses (negative loop). This model helps to explain both the specific phenotype and low frequencies of HCV specific CTL in chronic infection, through early elimination of cells before expansion and maturation can occur. The outcome of HCV infection is likely to result from the early balance between these two simultaneous loops.

Karrer U, Sierro S, Wagner M, Oxenius A, Hengel H, Koszinowski UH, Phillips RE, Klenerman P. 2003. Erratum: Memory inflation: Continous accumulation of antiviral CD8 + T cells over time (Journal of Immunology (2003) 170 (2022-2029)) Journal of Immunology, 171 (7), pp. 3895.

Cunliffe SL, Wyer JR, Sutton JK, Lucas M, Harcourt G, Klenerman P, McMichael AJ, Kelleher AD. 2002. Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells. Eur J Immunol, 32 (12), pp. 3366-3375. | Show Abstract | Read more

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.

Barnes E, Harcourt G, Brown D, Lucas M, Phillips R, Dusheiko G, Klenerman P. 2002. The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection. Hepatology, 36 (3), pp. 743-754. | Show Abstract | Read more

Hepatitis C virus (HCV) readily sets up a persistent infection and is a major cause of liver disease worldwide. Interferon alfa and ribavirin therapy lead to sustained clearance of virus in 31% to 64% of patients with type 1 and non-type 1 genotypes, respectively. It is not clear to what extent these drugs act directly to reduce HCV replication, or indirectly via host immune responses, and what evoked immune responses are associated with clinical outcome. We have examined prospectively 15 patients with chronic HCV infection before, during, and after combination therapy. Quantitative assays for HCV antigen-specific CD4+ and CD8+ T-cell responses, and flow cytometric assays for analysis of the phenotype of T cells, in addition to viral sequencing of core protein, were performed throughout the treatment and follow-up period over 18 months. We found enhancement of proliferative T-cell responses during therapy. Proliferative responses are strikingly heterogeneous in terms of specificity, kinetics, and magnitude. Proliferative responses are often not associated with interferon-gamma release. T-cell responses are rarely sustained irrespective of treatment outcome and this is not due to the evolution of new immune escape variants. T-cell responses tend to peak late in the course of treatment. In conclusion, combination therapy for HCV has a transient effect on host virus-specific T cells in the blood. Induction of sustained T-cell responses may require additional immune modulation later in therapy.

Hennig BJ, Hellier S, Frodsham AJ, Zhang L, Klenerman P, Knapp S, Wright M, Thomas HC, Thursz M, Hill AV. 2002. Association of low-density lipoprotein receptor polymorphisms and outcome of hepatitis C infection. Genes Immun, 3 (6), pp. 359-367. | Show Abstract | Read more

The low-density lipoprotein receptor (LDLR) has been proposed to promote hepatitis C virus endocytosis and the cell membrane protein CD81 may also promote HCV host cell entry. The CD81 gene was sequenced to screen for novel polymorphisms, but no SNPs were identified. Polymorphisms within the LDLR gene are associated with the pathogenesis of familial hypercholesterolemia, atherosclerosis and obesity. We therefore studied genetic variation within the LDLR gene and clinical features of hepatitis C infection. An amino acid change in exon 8 was associated with severity of fibrosis; a SNP in exon 10 correlated with viral clearance and overall inflammation, and a SNP in the 3'UTR appeared to influence treatment response. There were no other significant associations between any of the SNPs studied and the clinical measures of hepatitis C infection. We furthermore report on linkage disequilibrium within the gene and haplotype frequencies in our population. Our findings support a possible role for the LDLR in the modulation of disease progression by affecting immune responses, rather than functioning as receptor for HCV.

Klenerman P, Wu Y, Phillips R. 2002. HIV: current opinion in escapology. Curr Opin Microbiol, 5 (4), pp. 408-413. | Show Abstract | Read more

Much recent work strongly supports the hypothesis that CD8(+) T lymphocytes (CTLs) exert important immune control over HIV and so are a major selective force in its evolution. We analyse this host-pathogen interplay and focus on new data that describe the overall 'effectiveness' of CTL responses (strength, spread, specificity and 'stamina') and the mechanisms by which HIV may evade this suppressive activity. CTLs directed against HIV recognise very large numbers of distinct epitopes across the genome, are largely functional, turn over rapidly, and possess a phenotype that is distinct from CD8(+) lymphocytes specific for other viruses. Mutation of HIV epitopes that alters or abolishes CTL recognition altogether appears to be the most important immune escape mechanism, as the variation that HIV generates defies the limits of the T cell repertoire. However, this immune evasion is still only well-studied in a few patients. The rules that govern immune escape, and the ultimate limits of CTL capacity to deal with the variant epitopes that currently circulate, are not understood. This information will determine the feasibility of current vaccine approaches that, so far, make no provision for the enormous antigenic plasticity of HIV.

Lauer GM, Ouchi K, Chung RT, Nguyen TN, Day CL, Purkis DR, Reiser M, Kim AY, Lucas M, Klenerman P, Walker BD. 2002. Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities. J Virol, 76 (12), pp. 6104-6113. | Show Abstract | Read more

The hepatitis C virus (HCV)-specific CD8(+)-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8(+)-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8(+)-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8(+)-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.

Klenerman P, Tolfvenstam T, Price DA, Nixon DF, Broliden K, Oxenius A. 2002. T lymphocyte responses against human parvovirus B19: small virus, big response. Pathol Biol (Paris), 50 (5), pp. 317-325. | Show Abstract | Read more

Parvovirus B19 elicits both humoral and cellular immune responses. Recently some advances have been made in determining the frequencies, peptide targets and function of virus-specific CD8+ T lymphocyte responses. A single HLA B35-restricted epitope derived from the NS1 protein has been studied so far, but others clearly exist. Surprisingly large, persistent responses have been detected in healthy seropositive individuals, using interferon-gamma ELISpot assays and HLA class I peptide tetramers. Similar techniques are available for exploration of the CD4+ T cell epitopes, although less detail is currently available. Mapping of cellular immune responses against the entire B19 genome (the parvovirus "immunome") is now possible and if similarly large populations are found consistently, this could yield important insight into normal immunological control and abnormalities in B19-related disease.

McMichael A, Klenerman P. 2002. HIV/AIDS. HLA leaves its footprints on HIV. Science, 296 (5572), pp. 1410-1411. | Read more

Ward S, Lauer G, Isba R, Walker B, Klenerman P. 2002. Cellular immune responses against hepatitis C virus: the evidence base 2002. Clin Exp Immunol, 128 (2), pp. 195-203. | Show Abstract | Read more

Hepatitis C virus (HCV) is an RNA virus which is estimated to persistently infect about 170 million people worldwide. After acute infection, there is an initial period during which long-term outcome is decided. There is strong evidence that the cellular immune responses, involving both CD4+ and CD8+ T lymphocytes, are involved at this stage and it is their effectiveness which determines outcome. What is not understood is what determines their effectiveness. The most important component of this is likely to be some aspect of epitope selection, itself dictated by host MHC. Thus, to understand host immunity to HCV, we need to have a detailed understanding of the peptides involved in T lymphocyte responses. In this review, we discuss the peptide epitopes that have been identified so far, and their potential significance. We relate this to a scheme of host defence which may be useful for understanding natural and vaccine-induced immunity.

Klenerman P, Cerundolo V, Dunbar PR. 2002. Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol, 2 (4), pp. 263-272. | Show Abstract | Read more

To understand the success or failure of immune responses against pathogens or tumours requires the direct measurement of specific lymphocytes. Recently, there has been an explosion of data in this field through the use of several new tools for measuring the number and function of T cells. This has allowed immunologists who study human disease and mouse models of infection and cancer to readily track specific T cells--in both time and space. Although there are common patterns, over time, each host-pathogen relationship seems to develop unique characteristics, as reflected in the quality of the T-cell response.

Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, Ogg GS, King A et al. 2002. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med, 8 (4), pp. 379-385. | Show Abstract | Read more

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

Bonilla WV, Pinschewer DD, Klenerman P, Rousson V, Gaboli M, Pandolfi PP, Zinkernagel RM, Salvato MS, Hengartner H. 2002. Effects of promyelocytic leukemia protein on virus-host balance. J Virol, 76 (8), pp. 3810-3818. | Show Abstract | Read more

The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML(-/-) mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML(-/-) mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML(-/-) fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.

Lauer GM, Nguyen TN, Day CL, Robbins GK, Flynn T, McGowan K, Rosenberg ES, Lucas M, Klenerman P, Chung RT, Walker BD. 2002. Human immunodeficiency virus type 1-hepatitis C virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses. J Virol, 76 (6), pp. 2817-2826. | Show Abstract | Read more

Both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lead to chronic infection in a high percentage of persons, and an expanding epidemic of HIV-1-HCV coinfection has recently been identified. These individuals provide an opportunity for simultaneous assessment of immune responses to two viral infections associated with chronic plasma viremia. In this study we analyzed the breadth and magnitude of the CD8(+)- and CD4(+)-T-lymphocyte responses in 22 individuals infected with both HIV-1 and HCV. A CD8(+)-T-lymphocyte response against HIV-1 was readily detected in all subjects over a broad range of viral loads. In marked contrast, HCV-specific CD8(+)-T-lymphocyte responses were rarely detected, despite viral loads in plasma that were on average 1,000-fold higher. The few HCV-specific responses that were observed were relatively weak and limited in breadth. CD4-proliferative responses against HIV-1 were detected in about half of the coinfected subjects tested, but no proliferative response against any HCV protein was found in these coinfected persons. These data demonstrate a major discordance in immune responses to two persistent RNA viruses. In addition, they show a consistent and profound impairment in cellular immune responses to HCV compared to HIV-1 in HIV-1-HCV-coinfected persons.

Hunziker L, Klenerman P, Zinkernagel RM, Ehl S. 2002. Exhaustion of cytotoxic T cells during adoptive immunotherapy of virus carrier mice can be prevented by B cells or CD4+ T cells. Eur J Immunol, 32 (2), pp. 374-382. | Show Abstract | Read more

Rapid disappearance of antiviral CTL after transfusion into persistently infected individuals is a serious limitation of adoptive immunotherapy protocols. In the mouse model of persistent infection with lymphocytic choriomeningitis virus (LCMV) naive or immune virus-specific donor CD8+ T cells are exhausted after transfusion into carrier recipients with similar kinetics. Here we show that cotransfusion of immune CD4+ T cells prevents exhaustion of immune CD8+ T cells. Interestingly, cotransfer of primed B cells also prevented CD8+ T cell exhaustion in carriers even in the absence of T helper cells. This effect required the presence of immune B cells as repetitive treatment with hyperimmune serum led to the generation of antibody escape mutants. A combination of primed CD4+ T cells and primed B cells enhanced antiviral effects and prevented exhaustion also of naive CD8+ T cells. One key factor for prevention of CD8+ T cell exhaustion was the antiviral effect of the cotransfused cells thus reducing the time that CD8+ T cells are confronted with a high systemic viral load. These findings have implications for improving adoptive immunotherapy for persistent human viral infections.

Klenerman P, Lucas M, Barnes E, Harcourt G. 2002. Immunity to hepatitis C virus: stunned but not defeated. Microbes Infect, 4 (1), pp. 57-65. | Show Abstract | Read more

Hepatitis C virus (HCV) readily causes a persistent infection, although some individuals spontaneously control infection. 'Successful' immune responses appear to be multi-specific and sustained-including a major role for CD4(+)T cells. Some antiviral CD8(+)T cells show reduced capacity to secrete antiviral cytokines either temporarily ('stunning') or in the long term ('stunting'). The co-ordination of multiple immune effector functions may be required to gain control of HCV.

Barnes E, Lauer G, Walker B, Klenerman P. 2002. T cell failure in hepatitis C virus infection. Viral Immunol, 15 (2), pp. 285-293. | Read more

Pathan AA, Wilkinson KA, Klenerman P, McShane H, Davidson RN, Pasvol G, Hill AV, Lalvani A. 2001. Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J Immunol, 167 (9), pp. 5217-5225. | Show Abstract

The wide spectrum of clinical outcomes following infection with Mycobacterium tuberculosis is largely determined by the host immune response; therefore, we studied several clinically defined groups of individuals (n = 120) that differ in their ability to contain the bacillus. To quantitate M. tuberculosis-specific T cells directly ex vivo, we enumerated IFN-gamma-secreting CD4 T cells specific for ESAT-6, a secreted Ag that is highly specific for M. tuberculosis, and a target of protective immune responses in animal models. We found that frequencies of circulating ESAT-6 peptide-specific IFN-gamma-secreting CD4 T cells were higher in latently infected healthy contacts and subjects with minimal disease and low bacterial burdens than in patients with culture-positive active pulmonary tuberculosis (p = 0.009 and p = 0.002, respectively). Importantly, the frequency of these Ag-specific CD4 T cells fell progressively in all groups with treatment (p = 0.005), suggesting that the lower responses in patients with more extensive disease were not due to tuberculosis-induced immune suppression. This population of M. tuberculosis Ag-specific Th1-type CD4 T cells appears to correlate with clinical phenotype and declines during successful therapy; these features are consistent with a role for these T cells in the containment of M. tuberculosis in vivo. Such findings may assist in the design and evaluation of novel tuberculosis vaccine candidates.

Karrer U, Oxenius A, Phillips R, Klenerman P. 2001. Memory T cells: total recall or just a sense of déjà vu? Nat Immunol, 2 (11), pp. 991-993. | Read more

Lucas M, Karrer U, Lucas A, Klenerman P. 2001. Viral escape mechanisms--escapology taught by viruses. Int J Exp Pathol, 82 (5), pp. 269-286. | Show Abstract | Read more

Viruses have 'studied' immunology over millions of years of coevolution with their hosts. During this ongoing education they have developed countless mechanisms to escape from the host's immune system. To illustrate the most common strategies of viral immune escape we have focused on two murine models of persistent infection, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). LCMV is a fast replicating small RNA virus with a genome prone to mutations. Therefore, LCMV escapes from the immune system mainly by two strategies: 'speed' and 'shape change'. At the opposite extreme, MCMV is a large, complex DNA virus with a more rigid genome and thus the strategies used by LCMV are no option. However, MCMV has the coding capacity for additional genes which interfere specifically with the immune response of the host. These escape strategies have been described as 'camouflage' and 'sabotage'. Using these simple concepts we describe the spectrum of viral escapology, giving credit not only to the researchers who uncovered this fascinating area of immunology but also to the viruses themselves, who still have a few lessons to teach.

Lauer GM, Nguyen TN, Day CL, Klenerman P, Chung RT, Walker BD. 2001. Comprehensive analysis of the HCV-specific CD8+T cell response in individuals with chronic or resolved HCV infection. HEPATOLOGY, 34 (4), pp. 432A-432A.

Mullerova I, Rigopoulou E, Haigh P, Cooksley H, Lucas M, Klenerman P, Mesogiti D, Jenkins J, Williams R, Naoumov NV. 2001. Quantitative and qualitative changes in HCV-specific liver infiltrating T-cells parallel the progression of liver damage in chronic hepatitis C. HEPATOLOGY, 34 (4), pp. 430A-430A.

Vargas AL, Lechner F, Kantzanou M, Phillips RE, Klenerman P. 2001. Ex vivo analysis of phenotype and TCR usage in relation to CD45 isoform expression on cytomegalovirus-specific CD8+ T lymphocytes. Clin Exp Immunol, 125 (3), pp. 432-439. | Show Abstract | Read more

Human cytomegalovirus (CMV) is a ubiquitous pathogen which sets up a lifelong persistent infection and which can lead to significant disease in the immunosuppressed. The immunological mechanisms controlling CMV in the long term are not defined completely, but CD8+ T lymphocytes are thought to play an important role. Antiviral CD8+ T lymphocytes may exist in very large pools in healthy individuals. Although the detailed composition of these pools is not completely understood, there is known to be heterogeneity, in particular of CD45 isoform expression. We have therefore investigated the CD8+ T-lymphocyte response against CMV directly ex vivo using Class I tetramers combined with stains for a range of phenotypic markers followed by four-colour flow cytometric analysis. In particular, we examined expression of these phenotypic markers in relation to the expression of CD45 isoforms. We found that a spectrum of phenotypes exists stably, from CD45R0(high)/RA(low) through CD45RA(high)/R0(low), and that expression of other surface markers such as CD28 and CD62L, and also TCR usage, may vary in parallel with CD45 isoform expression. In some individuals, expansions of antigen-specific CD8+ T lymphocytes bearing specific TCR Vbeta chains were restricted to cells of particular CD45 isoforms. Immunity against CMV comprises a large population of CD8+ T lymphocytes with heterogeneous potential, a spectrum in which CD45 isoform expression may play a central role.

Luzyanina T, Engelborghs K, Ehl S, Klenerman P, Bocharov G. 2001. Low level viral persistence after infection with LCMV: a quantitative insight through numerical bifurcation analysis. Math Biosci, 173 (1), pp. 1-23. | Show Abstract | Read more

Many important viruses persist at very low levels in the body in the face of host immunity, and may influence the maintenance of this state of 'infection immunity'. To analyse low level viral persistence in quantitative terms, we use a mathematical model of antiviral cytotoxic T lymphocyte (CTL) response to lymphocytic choriomeningitis virus (LCMV). This model, described by a non-linear system of delay differential equations (DDEs), is studied using numerical bifurcation analysis techniques for DDEs. Domains where low level LCMV coexistence with CTL memory is possible, either as an equilibrium state or an oscillatory pattern, are identified in spaces of the model parameters characterising the interaction between virus and CTL populations. Our analysis suggests that the coexistence of replication competent virus below the conventional detection limit (of about 100 pfu per spleen) in the immune host as an equilibrium state requires the per day relative growth rate of the virus population to decrease at least 5-fold compared to the acute phase of infection. Oscillatory patterns in the dynamics of persisting LCMV and CTL memory, with virus population varying between 1 and 100 pfu per spleen, are possible within quite narrow intervals of the rates of virus growth and precursor CTL population death. Whereas the virus replication rate appears to determine the stability of the low level virus persistence, it does not affect the steady-state level of the viral population, except for very low values.

Grabowska AM, Lechner F, Klenerman P, Tighe PJ, Ryder S, Ball JK, Thomson BJ, Irving WL, Robins RA. 2001. Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection. Eur J Immunol, 31 (8), pp. 2388-2394. | Show Abstract | Read more

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV-infected patients and controls, focusing on the antigen-specific CD8(+) T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT-PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV-specific CD8(+) cells in the two compartments. T cell receptor usage in the liver of HCV-infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8(+) cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8(+) cells. A greater proportion of the liver tetramer-positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8(+) cells in the liver. In the course of chronic HCV infection, HCV-specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. 2001. A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis (vol 249, pg 99, 2001) JOURNAL OF IMMUNOLOGICAL METHODS, 252 (1-2), pp. 219-220. | Read more

Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, Walker B, Sullivan J, Phillips R, Pape GR, Klenerman P. 2001. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J Virol, 75 (12), pp. 5550-5558. | Show Abstract | Read more

Hepatitis C virus (HCV) sets up persistent infection in the majority of those exposed. It is likely that, as with other persistent viral infections, the efficacy of T-lymphocyte responses influences long-term outcome. However, little is known about the functional capacity of HCV-specific T-lymphocyte responses induced after acute infection. We investigated this by using major histocompatibility complex class I-peptide tetrameric complexes (tetramers), which allow direct detection of specific CD8+ T lymphocytes ex vivo, independently of function. Here we show that, early after infection, virus-specific CD8+ T lymphocytes detected with a panel of four such tetramers are abnormal in terms of their synthesis of antiviral cytokines and lytic activity. Furthermore, this phenotype is commonly maintained long term, since large sustained populations of HCV-specific CD8+ T lymphocytes were identified, which consistently had very poor antiviral cytokine responses as measured in vitro. Overall, HCV-specific CD8+ T lymphocytes show reduced synthesis of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) after stimulation with either mitogens or peptides, compared to responses to Epstein-Barr virus and/or cytomegalovirus. This behavior of antiviral CD8+ T lymphocytes induced after HCV infection may contribute to viral persistence through failure to effectively suppress viral replication.

Harcourt G, Hellier S, Bunce M, Satsangi J, Collier J, Chapman R, Phillips R, Klenerman P. 2001. Effect of HLA class II genotype on T helper lymphocyte responses and viral control in hepatitis C virus infection. J Viral Hepat, 8 (3), pp. 174-179. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is very common worldwide, but has a broad range of outcomes. A minority of patients are able to clear infection spontaneously, and this is thought to be due to the emergence and maintenance of effective cell-mediated immunity, particularly CD4+ T lymphocyte responses. Furthermore, genetic studies have indicated that HLA class II genotype strongly influences the outcome of infection. We have therefore investigated the influence of the protective HLA class II haplotype (DQB1*0301, which is in tight linkage disequilibrium with DRB1*1101) on the CD4+ T lymphocyte responses to HCV. We observe a strong association between this genotype and maintenance of a multispecific CD4+ T helper response. The effect on T helper responses was also maintained after combination interferon-alpha/ribavirin therapy, although the latter influenced the pattern of viral antigens to which patients responded. This is the first disease in which an association of HLA genotype with clinical outcome has been linked to an alteration of the immunological phenotype. The selection of protective peptides in those with the favourable HLA class II genotype may point in the direction of suitable vaccine candidates.

Karadimitris A, Gadola S, Altamirano M, Brown D, Woolfson A, Klenerman P, Chen JL, Koezuka Y et al. 2001. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc Natl Acad Sci U S A, 98 (6), pp. 3294-3298. | Show Abstract | Read more

CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipid specific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone- and foldase-assisted refolding of denatured CD1d molecules and beta(2)-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-alpha-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. 2001. A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis. J Immunol Methods, 249 (1-2), pp. 99-110. | Show Abstract | Read more

The 51Cr release assay has traditionally been used to investigate effector cell cytotoxic function against labeled targets, but this method has inherent problems that include hazards associated with radioactivity, cell labeling and high spontaneous release. Here we describe a novel flow cytometric assay which addresses and improves upon the problems currently encountered with the 51Cr release assay. The fluorometric assessment of T lymphocyte antigen specific lysis (FATAL) assay employs dual staining (PKH-26 and CFSE) to identify and evaluate the target population. We found that the PKH-26/CFSE combination efficiently labeled target cells. Evaluation of the spontaneous leakage from dye labeled target cells was forty fold lower than the spontaneous leakage seen with the 51Cr release assay. The FATAL assay permitted a more accurate assessment of the effector: target ratio, and detected low levels of cytotoxic T lymphocyte (CTL) mediated lysis. There was a strong correlation between the 51Cr release and FATAL assays, when performed in parallel with identical effector and target cells (r(2)=0.998, P=<0.0001). This novel method of detecting cytolysis represents a qualitative and quantitative improvement over standard 51Cr release analysis. The FATAL assay will be of value to further investigate mechanisms of cytolysis by effector cell populations.

Ciurea A, Hunziker L, Klenerman P, Hengartner H, Zinkernagel RM. 2001. Impairment of CD4(+) T cell responses during chronic virus infection prevents neutralizing antibody responses against virus escape mutants. J Exp Med, 193 (3), pp. 297-305. | Show Abstract | Read more

We have shown previously that neutralizing antibodies (nAbs) are important contributors to the long-term immune control of lymphocytic choriomeningitis virus infection, particularly if cytotoxic T cell responses are low or absent. Nevertheless, virus escape from the nAb response due to mutations within the surface glycoprotein gene may subsequently allow the virus to persist. Here we show that most of the antibody-escape viral mutants retain their immunogenicity. We present evidence that the failure of the infected host to mount effective humoral responses against emerging neutralization-escape mutants correlates with the rapid loss of CD4(+) T cell responsiveness during the establishment of viral persistence. Similar mechanisms may contribute to the persistence of some human pathogens such as hepatitis B and C viruses, and human immunodeficiency virus.

Bocharov G, Klenerman P, Ehl S. 2001. Predicting the dynamics of antiviral cytotoxic T-cell memory in response to different stimuli: cell population structure and protective function. Immunol Cell Biol, 79 (1), pp. 74-86. | Show Abstract | Read more

This paper examines the numerical and functional consequences of various stimuli on antiviral CD8+ T-cell memory using a mathematical model. The model is based upon biological evidence from the murine model of infection with lymphocytic choriomeningitis virus (LCMV) that the phenotype of immunological memory represents low-level responses driven by various stimuli, and the memory CTL population is partitioned between resting, cycling and effector cells. These subpopulations differ in their lifespan, their potential to mediate antiviral protection and in the stimuli needed for their maintenance. Three types of maintenance stimuli are examined: non-antigen-specific (bystander) stimulation, persisting antigen stimulation and reinfection-mediated stimulation. The modelling predicts that: (i) stable persistence of CTL memory requires the presence of either bystander or antigen-specific stimulation above a certain threshold depending on the sensitivity of memory CTL to stimulation and their life-span; (ii) a relatively low level of stimuli (approximately 10(4) fold less on a per CTL basis compared to acute infection) is needed to stabilize the expanded memory CTL population; (iii) the presence of CTL subsets in the memory pool of different activation states and lifespans ensures the robustness of memory persistence in the face of temporal variation in the low-level stimuli and; (iv) an 'optimal' population structure of the memory CTL pool, in terms of immediate protection, requires the presence of both activated cycling and effector CTL. For this, persisting antigen alone or synergistically with bystander signals provide the appropriate stimulation, so that the stimuli equivalent to approximately 30 p.f.u. of LCMV in the spleen are sufficient to maintain approximately 10(5)-10(6) specific CTL in the memory pool. These observations are relevant both to our understanding of natural protective immunity and to vaccine design.

Tolfvenstam T, Oxenius A, Price DA, Shacklett BL, Spiegel HM, Hedman K, Norbeck O, Levi M et al. 2001. Direct ex vivo measurement of CD8(+) T-lymphocyte responses to human parvovirus B19. J Virol, 75 (1), pp. 540-543. | Show Abstract | Read more

Parvovirus B19 is a common human pathogen which can cause severe syndromes, including aplastic anemia and fetal hydrops. The mapping of the first parvovirus B19-derived CD8(+) T-lymphocyte epitope is described. This HLA-B35-restricted peptide derives from the nonstructural (NS1) protein and is strongly immunogenic in B19 virus-seropositive donors.

Lin WR, Wozniak MA, Esiri MM, Klenerman P, Itzhaki RF. 2001. Herpes simplex encephalitis: involvement of apolipoprotein E genotype. J Neurol Neurosurg Psychiatry, 70 (1), pp. 117-119. | Show Abstract | Read more

It was previously found that herpes simplex type 1 virus (HSV1) when present in the brain, is a risk factor for Alzheimer's disease in carriers of the type 4 allele of the gene for apolipoprotein E (apoE epsilon4), and apoE epsilon4 is a risk factor for herpes labialis. Whether a specific allele of the gene is involved in susceptibility to another disorder caused by HSV1-herpes simplex encephalitis (HSE)-has now been investigated. DNA was prepared from formalin-fixed, paraffin-embedded blocks of specimens from the brain or spleen of 14 United Kingdom patients with HSE, confirmed by necropsy, and from the CSF of seven United Kingdom clinical patients with HSV1 in their CSF detected by polymerase chain reaction (PCR). ApoE genotype of the DNA from blocks was determined by seminested PCR, and of the DNA from CSF by one step PCR, followed by restriction endonuclease digestion. The apoE allele frequencies were compared with values previously obtained for 238 normal people from the United Kingdom. The apoE epsilon2 allele frequency of the patients with HSE was 26%, significantly higher than the value of 7% for the normal subjects (OR=4.6, 95% confidence interval (95% CI) 2. 0-10.8). The apoE epsilon3 and epsilon4 allele frequencies did not differ significantly between the two groups. Thus, it seems that apoE epsilon2 is a risk factor for HSE.

Lechner F, Cuero AL, Kantzanou M, Klenerman P. 2001. Studies of human antiviral CD8+ lymphocytes using class I peptide tetramers. Rev Med Virol, 11 (1), pp. 11-22. | Show Abstract | Read more

Understanding the interactions between a host and a pathogen relies crucially on quantitative measurements of immune responses. Until recently, measurements of the levels of cellular immune responses, i.e. those mediated by CD4+ and CD8+ T lymphocytes have depended largely on culture in vitro and subsequent measurement of specific functions (such as cytolysis). More recently, new technologies based around tetrameric class I peptide complexes (tetramers) have allowed immunologists to measure CD8+ T lymphocyte levels directly ex vivo and independently of function. Since CD8+ lymphocytes play a key role in a number of important human viral infections, these tools have yielded useful insights into the dynamics, phenotype and function of human antiviral lymphocyte populations. In this review we describe some of the basic aspects of the biology of virus-specific CD8+ lymphocytes, and the current methods available to detect them. The use of tetramers has, in just four years, transformed our understanding of the immune responses against HIV, HTLV-1, HBV, HCV, CMV and EBV, and holds promise in a number of areas where quantitative analysis of the antiviral response in terms of both number and function is critical.

Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. 2001. Erratum: A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis (Journal of Immunological Methods (2001) 249 (99-110) PII: S002217590000329X) Journal of Immunological Methods, 252 (1-2), pp. 219-220. | Read more

Barnes EJ, Harcourt G, Brown D, Phillips R, Geoffrey DM, Klenerman P. 2000. Hepatitis C virus (HCV) cellular immune responses are induced during combination therapy but are not sustained. HEPATOLOGY, 32 (4), pp. 227A-227A.

Gruener NH, Lechner F, Jung CM, Diepolder H, Gerlach TJ, Zachoval RZ, Schraut W, Schirren AC, Santantonio T, Klenerman P, Pape GR. 2000. Detection of HCV-specific CD8+T cells in acuter hepatitis C which are impaired in cytolytic function and production of cytokines. HEPATOLOGY, 32 (4), pp. 271A-271A.

Klenerman P, Lechner F, Kantzanou M, Ciurea A, Hengartner H, Zinkernagel R. 2000. Viral escape and the failure of cellular immune responses. Science, 289 (5487), pp. 2003. | Read more

Lechner F, Gruener NH, Urbani S, Uggeri J, Santantonio T, Kammer AR, Cerny A, Phillips R, Ferrari C, Pape GR, Klenerman P. 2000. CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained. Eur J Immunol, 30 (9), pp. 2479-2487. | Show Abstract | Read more

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

Lechner F, Sullivan J, Spiegel H, Nixon DF, Ferrari B, Davis A, Borkowsky B, Pollack H, Barnes E, Dusheiko G, Klenerman P. 2000. Why do cytotoxic T lymphocytes fail to eliminate hepatitis C virus? Lessons from studies using major histocompatibility complex class I peptide tetramers. Philos Trans R Soc Lond B Biol Sci, 355 (1400), pp. 1085-1092. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is a major public health problem, affecting an estimated 3% of the world's population, and over 10% in some countries. Infection in most cases becomes persistent, and can lead to hepatic inflammation, fibrosis and liver failure. The T lymphocyte reponse, in particular that mediated by cytotoxic T lymphocytes (CTLs), is likely to be involved in determining the outcome of infection, although its overall role is not clear. The use of major histocompatibility complex (MHC) class I peptide tetrameric complexes (tetramers) to study antiviral CTL responses has revolutionized our approach to the study of human infection. We have used a panel of MHC class I tetramers to analyse immune responses in HCV-infected individuals at various stages of disease. We find that the CTL response against HCV is vigorous in its early phases but dwindles over time both in terms of lymphocyte number and function. A number of potential explanations for this 'CTL failure' are discussed.

Goulder PJ, Lechner F, Klenerman P, McIntosh K, Walker BD. 2000. Characterization of a novel respiratory syncytial virus-specific human cytotoxic T-lymphocyte epitope. J Virol, 74 (16), pp. 7694-7697. | Show Abstract | Read more

Respiratory syncytial virus (RSV) infection is a major cause of morbidity in childhood worldwide. The first human RSV-specific cytotoxic T-lymphocyte epitope to be defined is described. This HLA B7-restricted epitope in nucleoprotein (NP) was detectable in four healthy, B7-positive adult subjects using B7-RSV-NP tetrameric complexes to stain CD8(+) T cells.

Hassan-Walker AF, Vargas AL, Mattes FM, Klenerman P, Lechner F, Burroughs AK, Griffiths PD, Phillips RE, Emery VC. 2000. Immunosuppression for liver transplantation with tacrolimus impairs specific cytomegalovirus CD8 immune responses CLINICAL INFECTIOUS DISEASES, 31 (1), pp. 317-317.

Seiler P, Senn BM, Klenerman P, Kalinke U, Hengartner H, Zinkernagel RM. 2000. Additive effect of neutralizing antibody and antiviral drug treatment in preventing virus escape and persistence. J Virol, 74 (13), pp. 5896-5901. | Show Abstract | Read more

Poorly cytopathic or noncytopathic viruses can escape immune surveillance and establish a chronic infection. Here we exploited the strategy of combining antiviral drug treatment with the induction of a neutralizing antibody response to avoid the appearance of neutralization-resistant virus variants. Despite the fact that H25 immunoglobulin transgenic mice infected with lymphocytic choriomeningitis virus mounted an early neutralizing antibody response, the virus escaped from neutralization and persisted. After ribavirin treatment of H25 transgenic mice, the appearance of neutralization-resistant virus was prevented and virus was cleared. Thus, the combination of virus-neutralizing antibodies and chemotherapy efficiently controlled the infection, whereas each defense line alone did not. Similar additive effects may be unexpectedly efficient and beneficial in humans after infections with persistent viruses such as hepatitis C virus and hepatitis B virus and possibly human immunodeficiency virus.

Lechner F, Wong DK, Dunbar PR, Chapman R, Chung RT, Dohrenwend P, Robbins G, Phillips R, Klenerman P, Walker BD. 2000. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med, 191 (9), pp. 1499-1512. | Show Abstract | Read more

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-gamma, a "stunned" phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

Barchet W, Oehen S, Klenerman P, Wodarz D, Bocharov G, Lloyd AL, Nowak MA, Hengartner H, Zinkernagel RM, Ehl S. 2000. Direct quantitation of rapid elimination of viral antigen-positive lymphocytes by antiviral CD8(+) T cells in vivo. Eur J Immunol, 30 (5), pp. 1356-1363. | Show Abstract | Read more

Lysis of infected cells by CD8(+) T cells is an important mechanism for the control of virus infections, but remains difficult to quantify in vivo. Here, we study the elimination kinetics of viral antigen-positive lymphocytes by antiviral CD8(+) T cells using flow cytometry and mathematical analysis. In mice acutely infected with lymphocytic choriomeningitis virus, more than 99.99 % of target cells were eliminated each day, corresponding to a half-life of 1.4 h. Even in mice exposed to virus 300 days previously, and with no ex vivo killing activity, 84 % of the target cells were eliminated per day. Unexpectedly, the elimination kinetics of antigen-positive lymphocytes was not significantly impaired in mice deficient in either perforin-, CD95 ligand- or TNF-mediated cytotoxicity. For viruses with a particular tropism for lymphocytes, such as Epstein-Barr virus or HIV, our results illustrate how effectively CD8(+) T cell-mediated elimination of target cells can potentially contribute to virus control and immunosuppression.

Hellier SC, Martin-Ginolhac M, Phillips RE, Chapman RW, Klenerman P. 2000. Do mutations in CD81 affect clinical outcome in patients with chronic hepatitis C infection? GASTROENTEROLOGY, 118 (4), pp. A939-A939.

Valiante NM, D'Andrea A, Crotta S, Lechner F, Klenerman P, Nuti S, Wack A, Abrignani S. 2000. Life, activation and death of intrahepatic lymphocytes in chronic hepatitis C. Immunol Rev, 174 pp. 77-89. | Show Abstract | Read more

The healthy liver of adult humans has little or no lymphocyte component and the histological finding of intrahepatic lymphocytes (IHL) is evidence of liver pathology. In a liver injured by chronic hepatitis C, the most common chronic liver disease, most IHL are activated/pro-inflammatory cells, which are particularly enriched for effectors of innate immunity (natural killer (NK), natural T, and other NK-like T cells). IHL do not undergo clonal expansion in the liver but migrate from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die, suggesting that maintenance of the IHL pool depends on continuous lymphocyte migration. The cytotoxic and inflammatory functions of these IHL have three potential outcomes: 1) they could be helpful in clearing the virus (a rare case in hepatitis C virus (HCV) infection); 2) they could be useless and have no effect on the infection; or 3) they could be harmful, whereby overaggressive lymphocyte responses destroy the liver in a continuous and unsuccessful attempt to clear the virus. Unfortunately, we do not know as of yet which of these possibilities is the case and, therefore, a more complete picture of the intrahepatic immune response will be relevant to the development of new therapeutic strategies against HCV. Additionally and from a more general perspective, due to the availability of biopsied material and the high prevalence (approximately 3%) of HCV infection worldwide, studying the chronically inflamed liver of hepatitis C patients is an ideal model to investigate the poorly understood processes of lymphocyte trafficking, activation and death to non-lymphoid sites of chronic inflammation in man.

Ciurea A, Klenerman P, Hunziker L, Horvath E, Senn BM, Ochsenbein AF, Hengartner H, Zinkernagel RM. 2000. Viral persistence in vivo through selection of neutralizing antibody-escape variants. Proc Natl Acad Sci U S A, 97 (6), pp. 2749-2754. | Show Abstract | Read more

Despite initial virus control by CD8(+) cytotoxic T lymphocytes (CTLs), noncytopathic or variably cytopathic viruses (e.g., hepatitis B and C viruses, HIV) are able to establish persistent infections. The role of neutralizing antibodies (nAbs) in controlling disease progression is unclear. Therefore, the phenomenon of viral evasion from the nAb response and its implications for virus persistence remain controversial. Here we demonstrate nAb-mediated viral clearance in CTL-deficient mice infected with the prototypic noncytopathic lymphocytic choriomeningitis virus (strain WE). During prolonged CTL absence, neutralization-resistant virus mutants were selected in individual mice within 70-90 days. In naive animals infected with these virus variants only low nAb responses were induced, resulting in an increased tendency of virus to persist.

Behboudi S, Chao D, Klenerman P, Austyn J. 2000. The effects of DNA containing CpG motif on dendritic cells. Immunology, 99 (3), pp. 361-366. | Show Abstract | Read more

Dendritic cells (DC) are specialized antigen-presenting cells. DC can acquire and process antigens in the periphery before maturing and migrating to secondary lymphoid tissues where they present the antigens and deliver co-stimulatory signals to T cells. We describe an immunostimulatory oligonucleotide containing a CpG motif that stimulated murine DC to up-regulate co-stimulatory molecules, induce T-cell proliferative responses and secrete interleukin-12 in vitro. Administration of this oligonucleotide, but not of a control oligonucleotide lacking this motif, to mice led to the disappearance of DC from the marginal zone and T-cell areas of spleen, but not from heart or kidney. The same CpG did not cause maturation of monocyte-derived human DC in vitro, but lipopolysaccharide-treated monocyte-derived DC showed enhanced functional activity and up-regulated co-stimulatory molecules.

Hellier S, Martin-Ginolhac M, Phiilips R, Chapman R, Klenerman P. 2000. Do mutations in CD 81 affect clinical outcome uv patients with chronic hepatitis C infection? JOURNAL OF HEPATOLOGY, 32 pp. 92-92. | Read more

Webster DP, Klenerman P, Dusheiko GM. 2015. Hepatitis C. Lancet, 385 (9973), pp. 1124-1135. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is a major health problem worldwide. The effects of chronic infection include cirrhosis, end-stage liver disease, and hepatocellular carcinoma. As a result of shared routes of transmission, co-infection with HIV is a substantial problem, and individuals infected with both viruses have poorer outcomes than do peers infected with one virus. No effective vaccine exists, although persistent HCV infection is potentially curable. The standard of care has been subcutaneous interferon alfa and oral ribavirin for 24-72 weeks. This treatment results in a sustained virological response in around 50% of individuals, and is complicated by clinically significant adverse events. In the past 10 years, advances in HCV cell culture have enabled an improved understanding of HCV virology, which has led to development of many new direct-acting antiviral drugs that target key components of virus replication. These direct-acting drugs allow for simplified and shortened treatments for HCV that can be given as oral regimens with increased tolerability and efficacy than interferon and ribavirin. Remaining obstacles include access to appropriate care and treatment, and development of a vaccine.

Nicoll G, Ni J, Liu D, Klenerman P, Munday J, Dubock S, Mattei MG, Crocker PR. 1999. Identification and characterization of a novel siglec, siglec-7, expressed by human natural killer cells and monocytes. J Biol Chem, 274 (48), pp. 34089-34095. | Show Abstract | Read more

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8(+) T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of approximately 65 kDa.

Ciurea A, Klenerman P, Hunziker L, Horvath E, Odermatt B, Ochsenbein AF, Hengartner H, Zinkernagel RM. 1999. Persistence of lymphocytic choriomeningitis virus at very low levels in immune mice. Proc Natl Acad Sci U S A, 96 (21), pp. 11964-11969. | Show Abstract | Read more

Lymphocytic choriomeningitis virus (LCMV), strain WE, is a non-cytopathic RNA virus that is highly adapted to its natural host, the mouse. Acute infection of adult mice leads to generalized virus spread, followed by cytotoxic T lymphocyte-mediated virus clearance below the detection levels of conventional assays within 2-3 weeks. Indirect evidence had suggested that virus or viral antigen might persist in the immune mouse. Here we demonstrate LCMV-WE persistence at low levels after infection with 10(2) or 10(6) plaque-forming units, shown as viral genome, viral antigen, and replicative virus using sensitive in vitro and in vivo assays. The finding that LCMV-WE persists in the face of apparently intact immune responses resembles the situation in some viral (hepatitis B and C, HIV) and bacterial (tuberculosis, leprosy) infections in humans; the results are relevant to the understanding not only of other murine and human persistent viral infections but also of protective immunological memory by "infection immunity."

Wong DKH, Lechner F, Dohrenwend PB, Chung R, Robbins G, Klenerman P, Walker BD. 1999. Dynamics of the CD8+and CD4+T cell immune response during acute HCV infection with spontaneous resolution. HEPATOLOGY, 30 (4), pp. 354A-354A.

Gruener NH, Lechner F, Gerlach TJ, Jung CM, Diepolder HM, Zachoval R, Schraut WW, Santanonio T, Klenerman P, Pape GR. 1999. Immune response in acute hepatitis C: Enumeration of virus specific CD8+T cells with ELISPOT assay and tetramers HEPATOLOGY, 30 (4), pp. 409A-409A.

Klenerman P, Zinkernagel RM. 1999. Of mice and men: cytotoxic T cells and AIDS pathogenesis. AIDS Read, 9 (7), pp. 474-480. | Show Abstract

CD8+ cytotoxic T lymphocytes (CTLs) represent a first line of defense against HIV infection, although their precise role in disease pathogenesis remains enigmatic. They play an important part in viral control but may also contribute to disease progression through destruction of CD4+ helper T cells. The role of CTLs in lymphocytic choriomeningitis virus (LCMV) infection in mice has been studied extensively, and the effects of CTL activity on host and virus are well defined. Although LCMV is not a retrovirus, it shares salient features with HIV, including a wide tropism, a capacity to persist, and genetic instability. The diseases caused by LCMV and HIV are linked by common immune effector mechanisms and, potentially, immunopathologies. Understanding the well-characterized immune responses in LCMV infection may therefore cast light on the role of CTLs in HIV disease.

Ochsenbein AF, Karrer U, Klenerman P, Althage A, Ciurea A, Shen H, Miller JF, Whitton JL, Hengartner H, Zinkernagel RM. 1999. A comparison of T cell memory against the same antigen induced by virus versus intracellular bacteria. Proc Natl Acad Sci U S A, 96 (16), pp. 9293-9298. | Show Abstract | Read more

Cytotoxic T cell (CTL) memory was analyzed after infection with lymphocytic choriomeningitis virus (LCMV) and recombinant Listeria monocytogenes (rLM) expressing the complete nucleoprotein of LCMV (rLM-NP(actA)) or only the immunodominant epitope of H-2(d) mice (rLM-NP(118-126)). Immunization with LCMV and rLM induced a long-lived increased CTL precursor (CTLp) frequency specific for the viral (NP(118-126)) and for the bacterial (LLO(91-99)) epitope, respectively. However, after infection with rLM memory, CTLs were less protective against an intravenous LCMV challenge infection than a comparable number of LCMV-induced memory T cells. LCMV, but not recombinant Listeria-induced memory T cells were able to protect against lethal choriomeningitis by LCMV or a subsequent peripheral infection with recombinant vaccinia virus expressing LCMV-NP. The protective memory after viral and after rLM immunization was paralleled by evidence of LCMV but not rLM antigen persistence on day 15 and 30 after vaccination. These results document a striking difference in protective T cell memory between viral and bacterial vaccines and indicate that rapid T cell-dependent immune protection correlates with antigen persistence.

Oxenius A, Martinic MM, Hengartner H, Klenerman P. 1999. CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines. J Virol, 73 (5), pp. 4120-4126. | Show Abstract

Synthetic nonmethylated oligonucleotides containing CpG dinucleotides (CpG-ODNs) have been shown to exhibit immunostimulatory activity. CpG-ODNs have the capacity to directly activate B cells, macrophages, and dendritic cells, and we show here that this is reflected by cell surface binding of oligonucleotides to these cell subsets. However, T cells are not directly activated by CpG-ODNs, which correlates with the failure to bind to the T-cell surface. Efficient competition for CpG-induced B-cell activation by non-CpG-containing oligonucleotides suggests that oligonucleotides might bind to an as yet undefined sequence-nonspecific receptor prior to cellular activation. Induction of protective T-cell responses against challenge infection with lymphocytic choriomeningitis virus (LCMV) or with recombinant vaccinia virus expressing the LCMV glycoprotein was achieved by immunizing mice with the immunodominant major histocompatibility complex class I-binding LCMV glycoprotein-derived peptide gp33 together with CpG-ODNs. In these experiments, B cells, potentially serving as CpG-ODN-activated antigen-presenting cells (APCs), were not required for induction of protective immunity since CpG-ODN-gp33-immunized B-cell-deficient mice were equally protected against challenge infection with both viruses. This finding suggested that macrophages and/or dendritic cells were sufficiently activated in vivo by CpG-ODNs to serve as potent APCs for the induction of naive T cells. Furthermore, treatment with CpG-ODN alone induced protection against infection with Listeria monocytogenes via antigen-independent activation of macrophages. These data suggest that CpG activation of macrophages and dendritic cells may provide a critical step in CpG-ODN adjuvant activity.

Price DA, Klenerman P, Booth BL, Phillips RE, Sewell AK. 1999. Cytotoxic T lymphocytes, chemokines and antiviral immunity. Immunol Today, 20 (5), pp. 212-216. | Show Abstract | Read more

Evidence that CD8+ CTLs produce chemokines following engagement of viral antigens, and that MIP-1alpha is required for an inflammatory response to virus challenge, suggests that these molecules are key elements in the generation of effective antiviral immunity. Here, David Price and colleagues argue that the antigen-dependent release of chemokines by CTLs provides an elegant mechanism linking localization, amplification and coordination of the antiviral immune response to specific recognition of infected host cells beyond the confines of the lymphoid system.

Zinkernagel RM, Planz O, Ehl S, Battegay M, Odermatt B, Klenerman P, Hengartner H. 1999. General and specific immunosuppression caused by antiviral T-cell responses. Immunol Rev, 168 (1), pp. 305-315. | Show Abstract | Read more

Immunosuppression caused by the non-cytopathic lymphocytic choriomeningitis virus (LCMV) (an RNA virus) is mediated by antiviral cytotoxic T cells that destroy LCMV-infected cells, also of the immune system. While this immunopathological destruction of antigen-presenting cells, macrophages and follicular dendritic cells and of some CD4+ T cells causes general immunosuppression and impairs immune response to third party antigens, it also enhances exhaustion/deletion of LCMV-specific CD8+ T-cell responses. LCMV seems in addition to infect neutralizing antibody-producing B cells via the specific receptor; immunopathological LCMV specific CD8+ T-cell-mediated elimination of these infected B cells (but not of uninfected internal virus antigen-specific B cells) causes a highly specific immunosuppression that delays neutralizing antibody responses and thereby enhances virus persistence. Both generalized and specific immunosuppression by CD8+ T-cell-mediated immunopathology may be involved in human infections with HIV, hepatitis B virus or hepatitis C virus.

Ochsenbein AF, Klenerman P, Karrer U, Ludewig B, Pericin M, Hengartner H, Zinkernagel RM. 1999. Immune surveillance against a solid tumor fails because of immunological ignorance. Proc Natl Acad Sci U S A, 96 (5), pp. 2233-2238. | Show Abstract | Read more

Many peripheral solid tumors such as sarcomas and carcinomas express tumor-specific antigens that can serve as targets for immune effector T cells. Nevertheless, overall immune surveillance against such tumors seems relatively inefficient. We studied immune surveillance against a s.c. sarcoma expressing a characterized viral tumor antigen. Surprisingly, the tumor cells were capable of inducing a protective cytotoxic T cell response if transferred as a single-cell suspension. However, if they were transplanted as small tumor pieces, tumors readily grew. Tumor growth correlated strictly with (i) failure of tumor cells to reach the draining lymph nodes and (ii) absence of primed cytotoxic T cells. Cytotoxic T cells were not tolerant or deleted because a tumor antigen-specific cytotoxic T cell response was readily induced in lymphoid tissue by immunization with virus or with tumor cells even in the presence of large tumors. Established tumors were rejected by vaccine-induced effector T cells if effector T cells were maintained by prolonged or repetitive vaccination, but not by single-dose vaccination. Thus, in addition to several other tumor-promoting parameters, some antigenic peripheral sarcomas-and probably carcinomas-may grow not because they anergize or tolerize tumor-specific T cells, but because such tumors are immunologically dealt with as if they were in a so-called immunologically privileged site and are ignored for too long.

André P, Groettrup M, Klenerman P, de Giuli R, Booth BL, Cerundolo V, Bonneville M, Jotereau F, Zinkernagel RM, Lotteau V. 1998. An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc Natl Acad Sci U S A, 95 (22), pp. 13120-13124. | Show Abstract | Read more

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Ehl S, Klenerman P, Zinkernagel RM, Bocharov G. 1998. The impact of variation in the number of CD8(+) T-cell precursors on the outcome of virus infection. Cell Immunol, 189 (1), pp. 67-73. | Show Abstract | Read more

We investigated the role of varying the initial number of naive antiviral CTL precursors on the dynamics of LCMV-DOCILE infection. C57BL/6 mice, exhibiting LCMV-specific CTLp frequencies of about 50, are protected against virus persistence over a range of infectious doses up to 10(4) pfu. With 10-fold higher doses, a 100-fold increase in CTLp is required to restore virus control. With doses above 10(6) pfu, elevation of the initial CTLp number leads only to lethal immunopathology. Similarly, a 1000-fold increase in the number of initial naïve CTLp enhances the overall kinetics of virus elimination, but cannot limit early virus spread within the first 48 h after low-dose infection (500 pfu). Increases in initial naïve virus-specific CTLp numbers are of limited benefit in antiviral control. In addition to the number of virus-specific T cells, the time period needed to reach cytolytic effector function is a limiting parameter.

Price DA, Meier UC, Klenerman P, Purbhoo MA, Phillips RE, Sewell AK. 1998. The influence of antigenic variation on cytotoxic T lymphocyte responses in HIV-1 infection. J Mol Med (Berl), 76 (10), pp. 699-708. | Show Abstract | Read more

The propensity of HIV-1 for genetic variation, a consequence of error-prone reverse transcription combined with high rates of replication, is thought to contribute to the establishment of persistent infection in the host despite the presence of a vigorous antiviral immune response. Protective immunity to viruses is mediated primarily by cytotoxic T lymphocytes, which recognize viral peptides of 8-11 amino acids bound to major histocompatibility complex class I molecules on the surface of infected cells. In this review we examine the mechanisms by which mutation within peptide antigen-encoding regions of the viral genome enables HIV-1 to evade recognition by virus-specific cytotoxic T lymphocytes. The discussion is relevant to other genetically unstable viruses and more generally to intracellular pathogens of variable antigenicity.

Klenerman P, Zinkernagel RM. 1998. Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature, 394 (6692), pp. 482-485. | Show Abstract | Read more

Some viruses, including human immunodeficiency virus (HIV) and hepatitis B virus (HBV) in humans, and lymphocytic choriomeningitis virus (LCMV) in mice, are initially controlled by cytotoxic T lymphocytes (CTLs), but may subsequently escape through mutation of the relevant T-cell epitope. Some of these mutations preserve the normal binding to major histocompatibility complex class I molecules, but present an altered surface to the T-cell antigen receptor. The exact role of these so-called altered peptide ligands in vivo is not clear. Here we report that mice primed with LCMV-WE strain respond to a subsequent infection by WE-derived CTL epitope variants with a CTL response directed against the initial epitope rather than against the new variant epitope. This phenomenon of 'original antigenic sin' was initially described in influenza and is an asymmetric pattern of protective antibody crossreactivity determined by exposure to previously existing strains, which may therefore extend to some CTL responses. Original antigenic sin by CTL leads to impaired clearance of variant viruses infecting the same individual and so may enhance the immune escape of mutant viruses evolving in an individual host.

Purbhoo MA, Sewell AK, Klenerman P, Goulder PJ, Hilyard KL, Bell JI, Jakobsen BK, Phillips RE. 1998. Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade. Proc Natl Acad Sci U S A, 95 (8), pp. 4527-4532. | Show Abstract | Read more

It is not known how human immunodeficiency virus type 1 (HIV-1)-derived antagonist peptides interfere with intracellular activation of cytotoxic T lymphocytes (CTL). We identified Gag epitope variants in HIV-1-infected patients that act as antagonists of CTL responses to unmutated epitopes. We then investigated the effect that presentation of each variant has on the early events of T cell receptor (TCR) signal transduction. We found that altered peptide ligands (APL) failed to induce phosphorylation of pp36, a crucial adaptor protein involved in TCR signal transduction. We further investigated the effect that simultaneous presentation of APL and native antigen at low, physiological, peptide concentrations (1 nM) has on TCR signal transduction, and we found that the presence of APL can completely inhibit induction of the protein tyrosine phosphorylation events of the TCR signal transduction cascade.

Wodarz D, Klenerman P, Nowak MA. 1998. Dynamics of cytotoxic T-lymphocyte exhaustion. Proc Biol Sci, 265 (1392), pp. 191-203. | Show Abstract | Read more

We examine simple mathematical models to investigate the circumstances under which the dynamics of cytotoxic T-lymphocyte (CTL) activation and differentiation may result in the loss of virus specific CD8+ cells, a process known as CTL exhaustion. We distinguish between two general classes of viruses: (i) viruses infecting cells that are not involved in the immune response; and (ii) viruses infecting antigen presenting cells (APCs) and helper cells. The models specify host and viral properties that lead to CTL exhaustion and indicate that this phenomenon is only likely to be observed with viruses infecting APCs and helper cells. Moreover, it is found that for such viruses, a high rate of replication and a low degree of cytopathogenicity promote the exhaustion of the CTL response. In addition, a high initial virus load and a low CD4+ cell count promote the occurrence of CTL exhaustion. These conclusions are discussed with reference to empirical data on lymphocytic choriomeningitis virus and on human immunodeficiency virus.

Ehl S, Klenerman P, Aichele P, Hengartner H, Zinkernagel RM. 1997. A functional and kinetic comparison of antiviral effector and memory cytotoxic T lymphocyte populations in vivo and in vitro. Eur J Immunol, 27 (12), pp. 3404-3413. | Show Abstract | Read more

To analyze the critical parameters for effective antiviral cytotoxic T lymphocyte (CTL) activity in vivo, control of lymphocytic choriomeningitis virus (LCMV) infection in the spleen was studied after adoptive transfer of different spleen cell populations into preinfected recipients. The quantitative, qualitative and kinetic requirements for virus control were defined and related to in vitro assays to compare the antiviral protective function of CTL from naive, acutely infected and memory mice. Treatment of mice with an established but limited LCMV infection by adoptive transfer of spleen cells from acutely LCMV-infected mice led to complete virus elimination mainly mediated by donor-derived CD8+ T cell-mediated, perforin-dependent cytotoxicity. Since virus is continuously spreading and the number of infected target cells rapidly increases, the time until target cell lysis is achieved was critical: if release of viral progeny was not prevented early, additional time to perform effector function did not improve overall virus control. When the function of various cell populations was compared in this model, we found that CTL from naive and memory mice perform considerably less well than CTL from acutely infected mice. In vitro studies indicated that this is probably due to the fact that they can not fulfill the limiting time requirements for immediate antiviral protection: while CTL from acutely infected mice can perform lytic effector function immediately, memory CTL require a considerable reactivation time before they can lyse infected target cells. This reactivation does not necessarily involve cell division. These findings illustrate how critical time limitations are for CTL to mediate early control of a dynamic virus infection in vivo.

Klenerman P, Hengartner H, Zinkernagel RM. 1997. A non-retroviral RNA virus persists in DNA form. Nature, 390 (6657), pp. 298-301. | Show Abstract | Read more

Infection of adult mice with lymphocytic choriomeningitis virus (LCMV), a non-cytopathic segmented RNA virus, leads initially to generalized infection, followed by clearance and subsequent life-long immunity. Indirect evidence has suggested that viral antigens may persist in lymphoid tissues during the phase of immunological memory, but viral genomic RNA has not been detected in previous studies. During a search for persistent virus in the spleen, we identified LCMV-specific sequences present as DNA by polymerase chain reaction (PCR) in mice over 200 days after infection. In vivo and in vitro studies revealed that reverse transcription of viral RNA into complementary DNA occurred after acute infection of cells of its natural hosts, mouse and hamster, but not of other species and could be inhibited in vitro by azidothymidine (AZT), indicating that this was mediated by endogenous reverse transcriptase activity. These findings reveal a surprising and new pathway of interaction between exogenous RNA viruses and endogenous retroviral, and perhaps other host components, that results in the persistence of virally determined DNA. We speculate that the latter may function in vivo as a form of DNA vaccine.

Klenerman P, Zinkernagel RM. 1997. What can we learn about human immunodeficiency virus infection from a study of lymphocytic choriomeningitis virus? Immunol Rev, 159 (1), pp. 5-16. | Show Abstract | Read more

The role of cytotoxic T lymphocytes (CTL) in human immunodeficiency virus (HIV) infection remains elusive. Since the discovery 10 years ago of high levels of specific CTL in this disease, some have argued that they play an important role in virus control, others that they drive disease progression through destruction of T helper cells, and others still that they play no obvious role at all. By contrast, the central role of CTL in murine lymphocytic choriomeningitis virus (LCMV) infection has been very clearly worked out through the use of in vivo depletion and adoptive transfer experiments, as well as knockout and transgenic mice. To interpret the possible roles for CTL in HIV, we have therefore made a comparison between what is known about CTL and their interaction with virus-infected cells in these two infections. This illustrates a potential critical role for these cells in both control of HIV replication and immune-mediated pathology, but one that is highly dependent on virus dose, distribution and dynamics.

Zinkernagel R, Klenerman P. 1997. Immune surveillance and AIDS progression. Curr Biol, 7 (7), pp. R403-R405.

Zinkernagel R, Klenerman P. 1997. Correspondence - Immune surveillance and AIDS progression CURRENT BIOLOGY, 7 (7), pp. R403-R404. | Read more

Price DA, Goulder PJ, Klenerman P, Sewell AK, Easterbrook PJ, Troop M, Bangham CR, Phillips RE. 1997. Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary infection. Proc Natl Acad Sci U S A, 94 (5), pp. 1890-1895. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) are thought to play a crucial role in the termination of the acute primary HIV-1 syndrome, but clear evidence for this presumption has been lacking. Here we demonstrate positive selection of HIV-1 proviral sequences encoding variants within a CTL epitope in Nef, a gene product critical for viral pathogenicity, during and after seroconversion. These positively selected HIV-1 variants carried epitope sequence changes that either diminished or escaped CTL recognition. Other proviruses had mutations that abolished the Nef epitope altogether. These results provide clear evidence that CTLs exert selection pressure on the viral population in acute HIV-1 infection.

Klenerman P, Phillips RE, Rinaldo CR, Wahl LM, Ogg G, May RM, McMichael AJ, Nowak MA. 1996. Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection. Proc Natl Acad Sci U S A, 93 (26), pp. 15323-15328. | Show Abstract | Read more

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells-at plausible peripheral blood mononuclear cell-to-target ratios-with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per microliter, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

Reid SW, McAdam S, Smith KJ, Klenerman P, O'Callaghan CA, Harlos K, Jakobsen BK, McMichael AJ, Bell JI, Stuart DI, Jones EY. 1996. Antagonist HIV-1 Gag peptides induce structural changes in HLA B8. J Exp Med, 184 (6), pp. 2279-2286. | Show Abstract | Read more

In the cellular immune response, recognition by CTL-TCRs of viral antigens presented as peptides by HLA class I molecules, triggers destruction of the virally infected cell (Townsend, A.R.M., J. Rothbard, F.M. Gotch, G. Bahadur, D. Wraith, and A.J. McMichael. 1986. Cell. 44:959-968). Altered peptide ligands (APLs) which antagonise CTL recognition of infected cells have been reported (Jameson, S.C., F.R. Carbone, and M.J. Bevan. 1993. J. Exp. Med. 177:1541-1550). In one example, lysis of antigen presenting cells by CTLs in response to recognition of an HLA B8-restricted HIV-1 P17 (aa 24-31) epitope can be inhibited by naturally occurring variants of this peptide, which act as TCR antagonists (Klenerman, P., S. Rowland Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, P. Giangrande, R.E. Phillips, and A. McMichael. 1994. Nature (Lond.). 369:403-407). We have characterised two CTL clones and a CTL line whose interactions with these variants of P17 (aa 24-31) exhibit a variety of responses. We have examined the high resolution crystal structures of four of these APLs in complex with HLA B8 to determine alterations in the shape, chemistry, and local flexibility of the TCR binding surface. The variant peptides cause changes in the recognition surface by three mechanisms: changes contributed directly by the peptide, effects transmitted to the exposed peptide surface, and induced effects on the exposed framework of the peptide binding groove. While the first two mechanisms frequently lead to antagonism, the third has more profound effects on TCR recognition.

Klenerman P, Luzzi G, McIntyre K, Phillips R, McMichael A. 1996. Identification of a novel HLA-A25-restricted epitope in a conserved region of p24 gag (positions 71-80). AIDS, 10 (3), pp. 348-350. | Read more

Klenerman P, Phillips R, McMichael A. 1996. Cytotoxic T-cell antagonism in HIV-1 SEMINARS IN VIROLOGY, 7 (1), pp. 31-39. | Show Abstract | Read more

The cytotoxic T-cell (CTL) response to human immunodeficiency virus Type 1 (HIV-1) is vigorous and sustained, but despite this, the virus persists. Natural variation arising within CTL epitopes may affect CTL recognition of infected targets and allow viral escape. Some of these variant epitopes appear to engage T-cell receptors but fail to activate the CTL normally. This can interfere with recognition of the unmutated epitope - a phenomenon known as T-cell antagonism. We discuss the evidence for this in HIV-1 using CTL and epitope variants derived from infected donors, and discuss its possible relevance in vivo.

Meier UC, Klenerman P, Griffin P, James W, Köppe B, Larder B, McMichael A, Phillips R. 1995. Cytotoxic T lymphocyte lysis inhibited by viable HIV mutants. Science, 270 (5240), pp. 1360-1362. | Show Abstract | Read more

Immune evasion by the human immunodeficiency virus (HIV) is unexplained but may involve the mutation of viral antigens. When cytotoxic T lymphocytes engaged CD4-positive cells that were acutely infected with HIV bearing natural variant epitopes in reverse transcriptase, substantial inhibition of specific antiviral lysis was observed. Mutant viruses capable of these transactive effects could facilitate the persistence of a broad range of HIV variants in the face of an active and specific immune response.

McAdam S, Klenerman P, Tussey L, Rowland-Jones S, Lalloo D, Phillips R, Edwards A, Giangrande P, Brown AL, Gotch F. 1995. Immunogenic HIV variant peptides that bind to HLA-B8 can fail to stimulate cytotoxic T lymphocyte responses. J Immunol, 155 (5), pp. 2729-2736. | Show Abstract

Cytotoxic T lymphocyte responses in HIV infection can be impaired through variation in the epitope regions of viral proteins such as a gag. We report here an analysis of variant epitope peptides in three gag epitopes presented by HLA B8. Fifteen variant peptides were examined for their binding to HLA-B8; all but one bound at concentrations comparable to known epitopes. All except two of those that bound could be recognized by CTL from an HLA-B8 positive HIV-1-infected patient and were therefore immunogenic. However, in a hemophiliac patient studied in detail, there was a failure to respond to two immunogenic peptide epitopes representing virus present as provirus in the patient's peripheral blood. In one case, the patient's CTL had previously responded to the peptide; in the other case, there was a good response to a peptide of closely related sequence. Thus there was a selective failure of the CTL response to some proviral epitopes. This impaired reaction to new variants could contribute to the loss of immune control of the infection.

Klenerman P, Meier UC, Phillips RE, McMichael AJ. 1995. The effects of natural altered peptide ligands on the whole blood cytotoxic T lymphocyte response to human immunodeficiency virus. Eur J Immunol, 25 (7), pp. 1927-1931. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTL) directed against human immunodeficiency virus (HIV)-1 are detectable in the majority of infected individuals, and their early appearance as the initial viremia is suppressed is thought to represent a potent antiviral response. Variation which arises in CTL epitopes can affect recognition by CTL, and we have observed previously that variant epitopes in HIV-1 gag which arise in HIV-1-seropositive donors may act as T cell receptor (TCR) antagonists of their own CTL (Klenerman et al., Nature 1994, 369: 403). The most important question arising from these observations is the extent of these immune escape mechanisms in vivo. Here we show that fresh, uncultured lymphocytes taken directly from HIV-1-infected patients are susceptible to TCR antagonism by variants present within their own virus. In contrast to HLA Class II-restricted T cell responses, where anergy may be induced, we find that in vitro, natural variants may stimulate and sustain growth of CTL. These CTL lines retain lytic specificity exclusively for the original peptide. If this represents events in vivo, natural HIV altered peptide ligands (APL) have the capacity to inhibit the range of CTL directed against an epitope, not simply those clones selected in vitro. Partial activation of CTL by APL could also act to drive an ineffectual CTL response incapable of lysing infected cells bearing these natural antigenic variants. Distortion of lymphocyte populations and function by APL might represent a further mechanism of immune evasion by HIV.

Nowak MA, May RM, Phillips RE, Rowland-Jones S, Lalloo DG, McAdam S, Klenerman P, Köppe B, Sigmund K, Bangham CR. 1995. Antigenic oscillations and shifting immunodominance in HIV-1 infections. Nature, 375 (6532), pp. 606-611. | Show Abstract | Read more

A typical protein antigen contains several epitopes that can be recognized by cytotoxic T lymphocytes (CTL), but in a characteristic antiviral immune response in vivo, CTL recognize only a small number of these potential epitopes, sometimes only one, this phenomenon is known as immunodominance. Antigenic variation within CTL epitopes has been demonstrated for the human immunodeficiency virus HIV-1 (ref. 11) and other viruses and such 'antigenic escape' may be responsible for viral persistence. Here we develop a new mathematical model that deals with the interaction between CTL and multiple epitopes of a genetically variable pathogen, and show that the nonlinear competition among CTL responses against different epitopes can explain immunodominance. This model suggests that an antigenically homogeneous pathogen population tends to induce a dominant response against a single epitope, whereas a heterogeneous pathogen population can stimulate complicated fluctuating responses against multiple epitopes. Antigenic variation in the immunodominant epitope can shift responses to weaker epitopes and thereby reduce immunological control of the pathogen population. These ideas are consistent with detailed longitudinal studies of CTL responses in HIV-1 infected patients. For vaccine design, the model suggests that the major response should be directed against conserved epitopes even if they are subdominant.

MCMICHAEL A, ROWLANDJONES S, KLENERMAN P, MCADAM S, GOTCH F, PHILLIPS R, NOWAK M. 1995. EPITOPE VARIATION AND T-CELL RECOGNITION JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 60-60.

LALLOO D, KLENERMAN P, MCADAM S, KOPPE B, ROWLANDJONES S, PHILLIPS R, MCMICHAEL A. 1995. IMMUNE ESCAPE IN HIV-INFECTION JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 292-292.

MCADAM S, KLENERMAN P, TUSSEY L, ROWLANDJONES S, LALLOO D, BROWN A, GOTCH F, MCMICHAEL A. 1995. IMMUNOGENIC HIV VARIANT PEPTIDES THAT BIND TO HLA-B8 BUT FAIL TO STIMULATE CYTOTOXIC T-LYMPHOCYTE RESPONSES JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 297-297.

KOPPE B, KLENERMAN P, MEIER U, LALLOO D, MCADAM S, ROWLANDJONES S, MCMICHAEL A, PHILLIPS R. 1995. VARIATION IN T-CELL EPITOPES OF HIV-1 - SEQUENCES DETECTED IN VIRAL-RNA JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 322-322.

Klenerman P, Rowland-Jones S, McAdam S, Edwards J, Daenke S, Lalloo D, Köppe B, Rosenberg W, Boyd D, Edwards A. 1994. Cytotoxic T-cell activity antagonized by naturally occurring HIV-1 Gag variants. Nature, 369 (6479), pp. 403-407. | Show Abstract | Read more

Most asymptomatic individuals infected with HIV-1 have a cytotoxic T lymphocyte (CTL) response to the virus Gag proteins which can be demonstrated in vitro. Epitopes have been mapped in p17 Gag and p24 Gag restricted by HLA-B8 (p17-3 and p24-13) and -B27 (p24-14). Viruses isolated from patients who make CTL responses to these peptides vary within the genetic sequences encoding these epitopes and some mutations lead to reduction in killing activity in vitro. This was attributed to either failure of the variant epitope to bind major histocompatibility complex class I or failure of T-cell receptors to bind the presented peptide. But peptide variants of class I-restricted epitopes cause 'antagonism', that is, the presence of a variant epitope (in the form of peptide) inhibits normal lysis of targets presenting the original epitope. This mirrors similar findings in class II-restricted systems. Here we report that naturally occurring variant forms of p17-3, p24-13 and p24-14 may cause antagonism of CTL lines derived from the same individuals. The effect is present if the epitopes are derived from synthetic peptides and when they are processed from full-length proteins expressed by either recombinant vaccinia constructs or replicating HIV.

Conlon CP, Klenerman P, Edwards A, Larder BA, Phillips RE. 1994. Heterosexual transmission of human immunodeficiency virus type 1 variants associated with zidovudine resistance. J Infect Dis, 169 (2), pp. 411-415. | Show Abstract | Read more

During zidovudine therapy, human immunodeficiency virus type 1 (HIV-1) acquires a distinctive set of mutations that diminish the sensitivity of the virus to this drug in vitro. An AIDS patient is described who, while being treated with zidovudine, transmitted HIV-1 bearing a drug resistance mutation to a young woman who had never received zidovudine treatment. DNA sequencing of HIV-1 proviruses confirmed that these 2 persons shared HIV genetic variants, including a mutation at codon 70 in the reverse transcriptase gene associated with reduced in vitro sensitivity to zidovudine. This mutation persisted in the woman > 1 year in the absence of antiretroviral therapy. HIV-1 with genetic markers of zidovudine resistance can be transmitted heterosexually, but it is uncertain whether dissemination of drug-resistant virus will substantially reduce the usefulness of this drug.

Luzzi GA, Brindle R, Sockett PN, Solera J, Klenerman P, Warrell DA. 1993. Brucellosis: imported and laboratory-acquired cases, and an overview of treatment trials. Trans R Soc Trop Med Hyg, 87 (2), pp. 138-141. | Show Abstract | Read more

Following the successful eradication of Brucella abortus infection in cattle, human brucellosis in England and Wales has become an uncommon imported disease. Culture of the organism presents a major laboratory hazard, and difficulties in identification may occur using a biochemical test-strip method. An overview of recent treatment trials of brucellosis indicates that regimens combining streptomycin and doxycycline are associated with a higher success rate (judged by the frequency of treatment failure and relapse following therapy) than combinations of rifampicin and doxycycline.

Klenerman P, Luzzi GA, Peto TE. 1993. Pneumococcal disease and HIV infection. Ann Intern Med, 118 (5), pp. 393-394. | Read more

Klenerman P, Sander JW, Shorvon SD. 1993. Mortality in patients with epilepsy: a study of patients in long term residential care. J Neurol Neurosurg Psychiatry, 56 (2), pp. 149-152. | Show Abstract | Read more

The causes of death in a group of patients with severe epilepsy in long term residential care over a period of 11 years were assessed and the standardised mortality rate (SMR) determined. A total of 3392 patient-years were surveyed. One hundred and thirteen deaths were recorded in the period and this represents an overall mortality rate which is almost twice the expected rate for this population (SMR = 1.9; 95% CI 1.6-2.3; p < 0.01). Most deaths were due to cancer (26%), bronchopneumonia (25%), circulatory diseases (24%), were seizure-related (12%) or due to sudden unexpected death (6%). The highest SMRs in the neoplasm sub-group were due to cancers of the pancreas (SMR = 6.2) and hepatobiliary tumours (SMR = 17.6). Twenty per cent of patients died of epilepsy or epilepsy related causes (that is accidents, during seizures, status or sudden unexpected death). One in every 480 patients died due to a sudden unexpected death. This study in a highly selected population seems to confirm suggestions that mortality rates are higher in patients with epilepsy than in the general population, but prospective studies are warranted to ascertain underlying mechanisms.

Klenerman P, Dickson H, Luzzi GA. 1992. Plasma lactate dehydrogenase estimation in the diagnosis of malaria. Ann Trop Med Parasitol, 86 (5), pp. 563-565.

Spiteri MA, Klenerman P, Sheppard MN, Padley S, Clark TJ, Newman-Taylor A. 1992. Seasonal cryptogenic organising pneumonia with biochemical cholestasis: a new clinical entity. Lancet, 340 (8814), pp. 281-284. | Show Abstract | Read more

The term cryptogenic organising pneumonia has been used for the combination of dyspnoea, cough, pleuritic pain, widespread shadows on chest radiographs, and histological evidence of intra-alveolar organisation with buds of granulation tissue within the alveoli. We report 12 patients with seasonal recurrence of this disorder for between 3 and 11 years. In all 12 patients, symptoms recurred between late February and early May every year, tending to increase in severity each year, and resolved between June and January. Chest radiography and computed tomography showed bilateral consolidation. Lung biopsy samples showed intra-alveolar buds of granulation tissue. There were many neutrophils within the lumina of medium-sized airways and terminal bronchioles showed evidence of obstruction by granulation tissue. Functionally, the predominant defect was restrictive and only 2 patients (life-long non-smokers) had airflow limitation. All 12 patients had very high activities of liver enzymes, suggesting intrahepatic cholestasis, but no other evidence of liver disease. Cultures of blood, sputum, lung tissue, and bronchoalveolar lavage fluid, viral screening, and complement fixation tests were consistently negative. In all patients all abnormalities responded rapidly to oral steroid therapy. These findings suggest a seasonal syndrome of organising pneumonia and biochemical abnormalities indicative of intrahepatic cholestasis. No aetiological factor has been identified, but the nature and periodicity of the illness point to an inhaled agent present in the environment for a limited period every year.

Klenerman P, Hammond C, Kulkarni VN, Mehta JM. 1991. [343] Vibration sense and tarsal disintegration Journal of Rehabilitation Research and Development, 28 (1), pp. 270-271.

Klenerman P, Hammond C, Kulkarni VN, Mehta JM. 1990. Vibration sense and tarsal disintegration. Indian J Lepr, 62 (4), pp. 422-428. | Show Abstract

The extent of loss of vibration and pressure sensations was assessed in 21 leprosy patients with disintegration of the tarsus. Feet which had and did not have tarsal disintegration both showed severe impairment of pressure sensation, but the loss of vibration sense was more severe in feet which had undergone the destructive process. It appears that loss of deep sensation is an important factor in the process of tarsal disintegration in feet which are already anaesthetic. Measurement of vibration sense using a biosthesiometer may be a valuable clinical test in the investigation and follow-up of the patient with the insensitive foot to identify those at risk of developing tarsal disintegration.

Taylor EG, Klenerman P. 1990. Acute renal failure after colloidal bismuth subcitrate overdose. Lancet, 335 (8690), pp. 670-671. | Read more

Cruickshank JK, Richardson JH, Morgan OS, Porter J, Klenerman P, Knight J, Newell AL, Rudge P, Dalgleish AG. 1990. Screening for prolonged incubation of HTLV-I infection in British and Jamaican relatives of British patients with tropical spastic paraparesis. BMJ, 300 (6720), pp. 300-304. | Show Abstract | Read more

OBJECTIVE: To compare the prevalence of antibody to and proviral DNA of the retrovirus HTLV-I in relatives of 11 British patients with tropical spastic paraparesis who had migrated from Jamaica before they developed symptoms, and to examine factors possibly related to transmission of HTLV-I. DESIGN: Migrant, family study. Antibody state was determined by several methods and confirmed by western blotting; the polymerase chain reaction was used to detect proviral DNA. SETTING: Britain and Jamaica. SUBJECTS: All available first degree relatives: those born and still resident in Jamaica (group 1); those born in Jamaica who migrated to Britain (group 2); and index patients' children who were born and resident in Britain (group 3). All had been breast fed and none had had blood transfusions. RESULTS: Of the 66 living relatives, 60 were traced. Seroprevalence among those born in Jamaica (irrespective of current residence) was 22% (10/46; 95% confidence limits 9 to 34%) compared with zero among British born offspring (0/14) and was higher in group 2 at 33% (7/21; 12 to 55%) than in group 1 at 12% (3/25; 0 to 25%). (Patients in group 1 had the greatest mean age.) Proviral DNA was not detected in any subject negative for HTLV-I antibody, making prolonged viral incubation in those negative for the antibody unlikely. CONCLUSION: In this sample factors related to place of birth and early residence were more important in transmission of HTLV-I than maternal or age effects. In areas with a low to moderate prevalence policies of preventing mothers who are carriers of the virus from breast feeding would be premature.

Klenerman P, Luzzi GA. 1990. Acyclovir and postherpetic neuralgia. Biomed Pharmacother, 44 (9), pp. 455-459. | Show Abstract | Read more

Studies have demonstrated the benefit of acyclovir, given intravenously or orally, on the acute illness in herpes zoster (HZ). Whether or not such treatment influences the subsequent development of postherpetic neuralgia (PHN) has been the subject of recent controversy. Intravenous acyclovir has not been shown to influence PHN significantly in prospective studies. Oral acyclovir in large doses may reduce PHN during the 3 months after acute HZ, but this effect has not been observed consistently in well-designed studies. From 3 months onwards, no trial has demonstrated a significant effect of oral acyclovir in reducing PHN. The way forward is discussed.

Klenerman P. 1989. Mosquitoes: how to be the perfect host. Int J Dermatol, 28 (6), pp. 370-372. | Read more

Klenerman P, Peto TE, Luzzi GA, Juel-Jensen BE. 1989. Antiviral treatment and postherpetic neuralgia. BMJ, 298 (6676), pp. 832. | Read more

Klenerman P. 1989. Prostaglandins and leprosy. A role for aspirin? Lepr Rev, 60 (1), pp. 51-58. | Show Abstract

Prostaglandins not only have a role in inflammation, but may also be involved as mediators in the immune response. Drugs which affect prostaglandin synthesis may therefore be potential tools with which to modulate disturbed immunity. These possibilities are discussed with reference to immunity in leprosy, and in particular reversal reactions.

Hammond CJ, Klenerman P. 1988. Protective sensation in the foot in leprosy. Lepr Rev, 59 (4), pp. 347-354.

Klenerman P, Hammond C. 1988. Vibration sensation in leprosy patches. Int J Lepr Other Mycobact Dis, 56 (3), pp. 466-468.

Klenerman P. 1987. Etiological factors in delayed-type hypersensitivity reactions in leprosy. Int J Lepr Other Mycobact Dis, 55 (4), pp. 702-712.

CARPENTER K, KLENERMAN P, BALL R, MITCHINSON M, CARPENTER T, HARRIS R, WILLIAMS D. 1985. PHYSICOCHEMICAL STUDIES OF HUMAN ATHEROMA ARCHIVES OF BIOLOGY, 96 (3), pp. 342-342.

Thomson E, Ip CL, Badhan A, Christiansen MT, Adamson W, Ansari MA, Bibby D, Breuer J et al. 2016. Comparison of next generation sequencing technologies for the comprehensive assessment of full-length hepatitis C viral genomes. J Clin Microbiol, pp. JCM.00330-16-JCM.00330-16. | Show Abstract | Read more

Affordable next generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting anti-viral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV viral sequence datasets and evaluated their utility for diagnostics and clinical assessment.NGS methods using (1) unselected HCV RNA (metagenomic); (2) pre-enrichment of HCV RNA by probe capture and (3) HCV pre-amplification by PCR implemented in four UK centres were compared. Metrics of sequence coverage and depth, quasispecies diversity and detection of DAA-resistance associated variants (RAVs), mixed HCV genotype and other co-infections were compared using a panel of samples of varying viral load, genotype and mixed HCV geno(sub)types. Each NGS method generated near complete genome sequences from over 90% of samples. Enrichment methods and PCR pre-amplification generated greater sequence depth and were more effective for low viral load samples. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus co-infections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing genotype and information on potential DAA resistance.

van Wilgenburg B, Scherwitzl I, Hutchinson EC, Leng T, Kurioka A, Kulicke C, de Lara C, Cole S et al. 2016. MAIT cells are activated during human viral infections. Nat Commun, 7 pp. 11653. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Hurst T, Pace M, Katzourakis A, Phillips R, Klenerman P, Frater J, Magiorkinis G. 2016. Human endogenous retrovirus (HERV) expression is not induced by treatment with the histone deacetylase (HDAC) inhibitors in cellular models of HIV-1 latency. Retrovirology, 13 (1), pp. 10. | Show Abstract | Read more

BACKGROUND: While antiretroviral therapies have improved life expectancy and reduced viral loads in HIV-1-positive individuals, the cessation of treatment results in a rebound of viral replication. This suggests that a reservoir of latently-infected cells remains within these patients, the identity of which is ill-defined and therefore difficult to target therapeutically. Current strategies are aimed at using drugs such as histone deacetylase (HDAC) inhibitors to induce the expression of latent HIV-1 proviruses in order to activate and ultimately eradicate this reservoir of infected cells. One concern with the use of HDAC inhibitors is that they could up-regulate human endogenous retroviruses (HERVs), as well as HIV-1, with potentially pathophysiological consequences. RESULTS: In this study, we analysed the transcription of HERV genes in HIV-1 latency T cell (J-LAT 8.4) and monocyte (U1) models following treatment with the HDAC inhibitors, vorinostat, panobinostat and romidepsin. We examined the expression of HERV-K (HML-2) env and pol, as well as the co-opted genes HERV-W env (syncytin-1), HERV-FRD env (syncytin-2), in these cell lines. Finally, we investigated HERV expression in primary human T cells. CONCLUSIONS: We show that HDAC inhibitors did not substantially increase the transcription of the analysed HERV env or pol genes, suggesting that histone acetylation is not crucial for controlling HERV expression in these experimental models and in ex vivo primary human T cells. Importantly, this indicates that unwanted HERV expression does not appear to be a barrier to the use of HDAC inhibitors in HIV-1 cure strategies.

Llibre A, López-Macías C, Marafioti T, Mehta H, Partridge A, Kanzig C, Rivellese F, Galson JD et al. 2016. LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation. J Immunol, 196 (5), pp. 2085-2094. | Show Abstract | Read more

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans.

Ussher JE, Phalora P, Cosgrove C, Hannaway RF, Rauch A, Günthard HF, Goulder P, Phillips RE, Willberg CB, Klenerman P. 2015. Molecular Analyses Define Vα7.2-Jα33+ MAIT Cell Depletion in HIV Infection: A Case-Control Study. Medicine (Baltimore), 94 (29), pp. e1134. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are an abundant antibacterial innate-like lymphocyte population. There are conflicting reports as to their fate in HIV infection. The objective of this study was to determine whether MAIT cells are truly depleted in HIV infection. In this case-control study of HIV-positive patients and healthy controls, quantitative real-time polymerase chain reaction was used to assess the abundance of messenger RNA (mRNA) and genomic DNA (gDNA) encoding the canonical MAIT cell T cell receptor (Vα7.2-Jα33). Comparison was made with flow cytometry. Significant depletion of both Vα7.2-Jα33 mRNA and gDNA was seen in HIV infection. Depletion of Vα7.2+CD161++ T cells was confirmed by flow cytometry. In HIV infection, the abundance of Vα7.2-Jα33 mRNA correlated most strongly with the frequency of Vα7.2+CD161++ cells. No increase was observed in the frequency of Vα7.2+CD161- cells among CD3+CD4- lymphocytes. MAIT cells are depleted from blood in HIV infection as confirmed by independent assays. Significant accumulation of a CD161- MAIT cell population is unlikely. Molecular approaches represent a suitable alternative to flow cytometry-based assays for tracking of MAIT cells in HIV and other settings.

Fergusson JR, Smith KE, Fleming VM, Rajoriya N, Newell EW, Simmons R, Marchi E, Björkander S et al. 2014. CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages. Cell Rep, 9 (3), pp. 1075-1088. | Show Abstract | Read more

The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage.

Swadling L, Capone S, Antrobus RD, Brown A, Richardson R, Newell EW, Halliday J, Kelly C et al. 2014. A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory. Sci Transl Med, 6 (261), pp. 261ra153. | Show Abstract | Read more

A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in na