register interest

Professor John Frater

Research Area: Immunology
Technology Exchange: Bioinformatics, Cell sorting, Cellular immunology, Flow cytometry and Vaccine production and evaluation
Scientific Themes: Immunology & Infectious Disease
Keywords: HIV, AIDS, Immune response, HLA Class I, vaccine and viral fitness
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It is increasingly apparent that Highly Active Antiretroviral Therapy (HAART) may not be the long-term solution to the management of HIV infection. Despite major improvements in morbidity and mortality in HIV+ve individuals, lifespan on HAART is less than for the HIV uninfected, there is unexplained morbidity on HAART (cardiovascular, renal, hepatic complications plus osteoporosis, dementia, ‘frailty’ and ageing), not all of which can explained by drug toxicities. In addition, the practicalities of global drug provision are currently untenable – achieving the US target of 80% HAART coverage in developing regions would account for half of the US foreign aid budget by 2016. Achieving a ‘sterilising cure’ (infectious disease model) or ‘drug-free remission’ (tumour model) must therefore become a priority for global HIV management.

There are fundamental issues impacting eradication, related to a persistent proviral HIV reservoir. Stopping HAART results in rebound of viraemia to levels similar to those recorded pre-therapy, HIV DNA can be detected in PBMCs and lymphoid tissue at all stages of therapy, the cellular and anatomical HIV reservoirs are not defined, there is no ‘undetectable viral load’ on HAART (median value is 3.1 copies/ml) and we do not fully understand the processes by which latency is induced, or how transcription is activated.

The CHERUB co-operative (Collaborative HIV Eradication of Reservoirs: UK BRC) is a NIHR-funded platform funded to explore strategies for achieving HIV eradication in the UK. CHERUB comprises internationally recognised researchers from Oxford, Cambridge, Imperial College, UCL and King’s College, and the associated NHS Trusts. Through CHERUB, we are recruiting new patient cohorts to allow studies varying from basic laboratory research, to ex vivo analyses, to large-scale clinical trials of novel interventions.

The role of Oxford University within CHERUB is to lead the scientific strategy (John Frater is co-PI and Scientific Lead). Specifically, our work will focus on quantification of viral reservoirs, sequence analysis by standard and next generation models, immunology (predominantly looking at the role of CTL in remission strategies) and more fundamental research into new approaches to eradication such as nanotechnology. Working in collaboration with NHS Trusts and also with the pharmaceutical industry, we aim to push forward the boundaries in this new and exciting field.

Name Department Institution Country
Jonathan Weber Imperial College, London United Kingdom
Sarah Fidler Imperial College United Kingdom
Professor Angela McLean Department of Zoology University of Oxford United Kingdom
Cloete van Vuuren University of Free State South Africa
The CHERUB Collaboration NIHR United Kingdom
Professor Rodney E Phillips Experimental Medicine Division University of Oxford United Kingdom
Professor Una O'Doherty University of Pennsylvania United States
Professor Sharon Lewin Infectious Diseases Monash University and Alfred Hospital Australia
Professor Helen McShane Jenner Institute University of Oxford United Kingdom
Julie Fox Guy's and St Thomas' NHS Trust United Kingdom
Kholoud Porter MRC Clinical Trials Unit United Kingdom

Hurst J, Hoffmann M, Pace M, Williams JP, Thornhill J, Hamlyn E, Meyerowitz J, Willberg C et al. 2015. Immunological biomarkers predict HIV-1 viral rebound after treatment interruption. Nat Commun, 6 pp. 8495. Read abstract | Read more

Treatment of HIV-1 infection with antiretroviral therapy (ART) in the weeks following transmission may induce a state of 'post-treatment control' (PTC) in some patients, in whom viraemia remains undetectable when ART is stopped. Explaining PTC could help our understanding of the processes that maintain viral persistence. Here we show that immunological biomarkers can predict time to viral rebound after stopping ART by analysing data from a randomized study of primary HIV-1 infection incorporating a treatment interruption (TI) after 48 weeks of ART (the SPARTAC trial). T-cell exhaustion markers PD-1, Tim-3 and Lag-3 measured prior to ART strongly predict time to the return of viraemia. These data indicate that T-cell exhaustion markers may identify those latently infected cells with a higher proclivity to viral transcription. Our results may open new avenues for understanding the mechanisms underlying PTC, and eventually HIV-1 eradication. Hide abstract

Adland E, Paioni P, Thobakgale C, Laker L, Mori L, Muenchhoff M, Csala A, Clapson M et al. 2015. Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection. PLoS Pathog, 11 (6), pp. e1004954. Read abstract | Read more

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression. Hide abstract

Frater J. 2015. Thirty years of treating HIV-1 infection: where next? Trans R Soc Trop Med Hyg, 109 (4), pp. 229-230. | Read more

Roberts HE, Hurst J, Robinson N, Brown H, Flanagan P, Vass L, Fidler S, Weber J et al. 2015. Structured observations reveal slow HIV-1 CTL escape. PLoS Genet, 11 (2), pp. e1004914. Read abstract | Read more

The existence of viral variants that escape from the selection pressures imposed by cytotoxic T-lymphocytes (CTLs) in HIV-1 infection is well documented, but it is unclear when they arise, with reported measures of the time to escape in individuals ranging from days to years. A study of participants enrolled in the SPARTAC (Short Pulse Anti-Retroviral Therapy at HIV Seroconversion) clinical trial allowed direct observation of the evolution of CTL escape variants in 125 adults with primary HIV-1 infection observed for up to three years. Patient HLA-type, longitudinal CD8+ T-cell responses measured by IFN-γ ELISpot and longitudinal HIV-1 gag, pol, and nef sequence data were used to study the timing and prevalence of CTL escape in the participants whilst untreated. Results showed that sequence variation within CTL epitopes at the first time point (within six months of the estimated date of seroconversion) was consistent with most mutations being transmitted in the infecting viral strain rather than with escape arising within the first few weeks of infection. Escape arose throughout the first three years of infection, but slowly and steadily. Approximately one third of patients did not drive any new escape in an HLA-restricted epitope in just under two years. Patients driving several escape mutations during these two years were rare and the median and modal numbers of new escape events in each patient were one and zero respectively. Survival analysis of time to escape found that possession of a protective HLA type significantly reduced time to first escape in a patient (p = 0.01), and epitopes escaped faster in the face of a measurable CD8+ ELISpot response (p = 0.001). However, even in an HLA matched host who mounted a measurable, specific, CD8+ response the average time before the targeted epitope evolved an escape mutation was longer than two years. Hide abstract

Thornhill J, Fidler S, Frater J. 2015. Advancing the HIV cure agenda: the next 5 years. Curr Opin Infect Dis, 28 (1), pp. 1-9. Read abstract | Read more

PURPOSE OF REVIEW: To explore how ethical considerations, improved diagnostics and data from clinical trials might see the lowering of some of the barriers blocking a cure for HIV infection over the next 5 years. RECENT FINDINGS: Despite the recent well publicized but eventually disappointing case reports, there remains only one successful HIV cure, the 'Berlin patient'. We will review the data suggesting that more potent agents might achieve in-vivo viral activation and explore the tantalizing phenomenon of 'posttreatment control' following treatment in primary HIV infection. We will also explore how new assays and novel interventions might move the field forward. SUMMARY: There is a need for new agents that can be safely tested to impact the viral reservoir, a more meaningful understanding of how to assay patient samples, and research into mechanisms behind how the reservoir is established and impacted by therapy. With HIV+ve individuals responding so well to antiretroviral therapy, new trials must be tested hand-in-hand with guidance from patient representatives, especially with respect to determining the acceptable risk. The road to a cure is going to be difficult, but it is vital that inevitable disappointments do not detract from the final goal, which remains worth striving for. Hide abstract

Williams JP, Hurst J, Stöhr W, Robinson N, Brown H, Fisher M, Kinloch S, Cooper D et al. 2014. HIV-1 DNA predicts disease progression and post-treatment virological control. Elife, 3 pp. e03821. Read abstract | Read more

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Hide abstract

Carlson JM, Schaefer M, Monaco DC, Batorsky R, Claiborne DT, Prince J, Deymier MJ, Ende ZS et al. 2014. Selection bias at the heterosexual HIV-1 transmission bottleneck SCIENCE, 345 (6193), pp. 1254031-1254031. Read abstract | Read more

Heterosexual transmission of HIV-1 typically results in one genetic variant establishing systemic infection.We compared, for 137 linked transmission pairs, the amino acid sequences encoded by non-envelope genes of viruses in both partners and demonstrate a selection bias for transmission of residues that are predicted to confer increased in vivo fitness on viruses in the newly infected, immunologically naïve recipient. Although tempered by transmission risk factors, such as donor viral load, genital inflammation, and recipient gender, this selection bias provides an overall transmission advantage for viral quasispecies that are dominated by viruses with high in vivo fitness. Thus, preventative or therapeutic approaches that even marginally reduce viral fitness may lower the overall transmission rates and offer long-term benefits even upon successful transmission. Hide abstract

Payne RP, Branch S, Kløverpris H, Matthews PC, Koofhethile CK, Strong T, Adland E, Leitman E et al. 2014. Differential escape patterns within the dominant HLA-B*57:03-restricted HIV Gag epitope reflect distinct clade-specific functional constraints. J Virol, 88 (9), pp. 4668-4678. Read abstract | Read more

UNLABELLED: HLA-B*57:01 and HLA-B*57:03, the most prevalent HLA-B*57 subtypes in Caucasian and African populations, respectively, are the HLA alleles most protective against HIV disease progression. Understanding the mechanisms underlying this immune control is of critical importance, yet they remain unclear. Unexplained differences are observed in the impact of the dominant cytotoxic T lymphocyte (CTL) response restricted by HLA-B*57:01 and HLA-B*57:03 in chronic infection on the Gag epitope KAFSPEVIPMF (KF11; Gag 162 to 172). We previously showed that the HLA-B*57:03-KF11 response is associated with a >1-log-lower viral setpoint in C clade virus infection and that this response selects escape mutants within the epitope. We first examined the relationship of KF11 responses in B clade virus-infected subjects with HLA-B*57:01 to immune control and observed that a detectable KF11 response was associated with a >1-log-higher viral load (P = 0.02). No evidence of HLA-B*57:01-KF11-associated selection pressure was identified in previous comprehensive analyses of >1,800 B clade virus-infected subjects. We then studied a B clade virus-infected cohort in Barbados, where HLA-B*57:03 is highly prevalent. In contrast to findings for B clade virus-infected subjects expressing HLA-B*57:01, we observed strong selection pressure driven by the HLA-B*57:03-KF11 response for the escape mutation S173T. This mutation reduces recognition of virus-infected cells by HLA-B*57:03-KF11 CTLs and is associated with a >1-log increase in viral load in HLA-B*57:03-positive subjects (P = 0.009). We demonstrate functional constraints imposed by HIV clade relating to the residue at Gag 173 that explain the differential clade-specific escape patterns in HLA-B*57:03 subjects. Further studies are needed to evaluate the role of the KF11 response in HLA-B*57:01-associated HIV disease protection. IMPORTANCE: HLA-B*57 is the HLA class I molecule that affords the greatest protection against disease progression in HIV infection. Understanding the key mechanism(s) underlying immunosuppression of HIV is of importance in guiding therapeutic and vaccine-related approaches to improve the levels of HIV control occurring in nature. Numerous mechanisms have been proposed to explain the HLA associations with differential HIV disease outcome, but no consensus exists. These studies focus on two subtypes of HLA-B*57 prevalent in Caucasian and African populations, HLA-B*57:01 and HLA-B*57:03, respectively. These alleles appear equally protective against HIV disease progression. The CTL epitopes presented are in many cases identical, and the dominant response in chronic infection in each case is to the Gag epitope KF11. However, there the similarity ends. This study sought to better understand the reasons for these differences and what they teach us about which immune responses contribute to immune control of HIV infection. Hide abstract

Jones M, Williams J, Gärtner K, Phillips R, Hurst J, Frater J. 2014. Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'. J Virol Methods, 202 pp. 46-53. Read abstract | Read more

Droplet Digital PCR (ddPCR) represents a new and alternative platform to conventional quantitative-PCR (qPCR) for the quantitation of DNA templates. However, the proposed improvement in sensitivity and reproducibility offered by ddPCR is not yet fully proven, partly because the delineation between positive and negative responses is not always clear. Data are presented demonstrating the sensitivity of the ddPCR system to both reagent concentrations and choice of cut-off for defining positive and negative results. By implementing k-nearest clustering, cut-offs are produced that improve the accuracy of ddPCR where target DNA is present at low copy numbers, a key application of ddPCR. This approach is applied to human albumin and HIV-1 proviral DNA ddPCR quantitative protocols. This tool is coded in JavaScript and has been made available for free in a web browser at Optimisation of the analyses of raw ddPCR data using 'definetherain' indicates that low target number detection can be improved by its implementation. Further application to patient samples will help define the clinical utility of this approach. Hide abstract

Pace M, Frater J. 2014. A cure for HIV: is it in sight? Expert Rev Anti Infect Ther, 12 (7), pp. 783-791. Read abstract | Read more

HIV is a devastating disease affecting millions of people worldwide despite the advent of successful antiretroviral therapy (ART). However, ART does not result in a cure and has to be taken for life. Accordingly, researchers are turning towards cure efforts, particularly in the light of two patients whose HIV has been seemingly eradicated. Numerous approaches and strategies have been considered for curing HIV, but no scalable and safe solution has yet been reached. With newly discovered difficulties in measuring the HIV reservoir, the main barrier to a cure, the only true test of cure is to stop ART and see whether the virus becomes detectable. However, it is possible that this treatment interruption may be associated with certain risks for patients. Here, we compare the current major approaches and recent advances for curing HIV, as well as discuss ways of evaluating HIV cure and the safety concerns involved. Hide abstract

Duncan CJ, Williams JP, Schiffner T, Gärtner K, Ochsenbauer C, Kappes J, Russell RA, Frater J, Sattentau QJ. 2014. High-multiplicity HIV-1 infection and neutralizing antibody evasion mediated by the macrophage-T cell virological synapse. J Virol, 88 (4), pp. 2025-2034. Read abstract | Read more

Macrophage infection is considered to play an important role in HIV-1 pathogenesis and persistence. Using a primary cell-based coculture model, we show that monocyte-derived macrophages (MDM) efficiently transmit a high-multiplicity HIV-1 infection to autologous CD4(+) T cells through a viral envelope glycoprotein (Env) receptor- and actin-dependent virological synapse (VS), facilitated by interactions between ICAM-1 and LFA-1. Virological synapse (VS)-mediated transmission by MDM results in high levels of T cell HIV-1 integration and is 1 to 2 orders of magnitude more efficient than cell-free infection. This mode of cell-to-cell transmission is broadly susceptible to the activity of CD4 binding site (CD4bs) and glycan or glycopeptide epitope-specific broadly neutralizing monoclonal antibodies (bNMAbs) but shows resistance to bNMAbs targeting the Env gp41 subunit membrane-proximal external region (MPER). These data define for the first time the structure and function of the macrophage-to-T cell VS and have important implications for bNMAb activity in HIV-1 prophylaxis and therapy. IMPORTANCE The ability of HIV-1 to move directly between contacting immune cells allows efficient viral dissemination with the potential to evade antibody attack. Here, we show that HIV-1 spreads from infected macrophages to T cells via a structure called a virological synapse that maintains extended contact between the two cell types, allowing transfer of multiple infectious events to the T cell. This process allows the virus to avoid neutralization by a class of antibody targeting the gp41 subunit of the envelope glycoproteins. These results have implications for viral spread in vivo and the specificities of neutralizing antibody elicited by antibody-based vaccines. Hide abstract

Frater J, Ewings F, Hurst J, Brown H, Robinson N, Fidler S, Babiker A, Weber J, Porter K, Phillips RE, SPARTAC Trial Investigators. 2014. HIV-1-specific CD4(+) responses in primary HIV-1 infection predict disease progression. AIDS, 28 (5), pp. 699-708. Read abstract | Read more

OBJECTIVES: Immune factors determining clinical progression following HIV-1 infection remain unclear. The SPARTAC trial randomized 366 participants in primary HIV infection (PHI) to different short-course therapies. The aim of this study was to investigate how early immune responses in PHI impacted clinical progression in SPARTAC. DESIGN AND METHODS: Participants with PHI recruited to the SPARTAC trial were sampled at enrolment, prior to commencing any therapy. HIV-1-specific CD4(+) and CD8(+) ELISpot responses were measured by gamma interferon ELISPOT. Immunological data were associated with baseline covariates and times to clinical progression using logistic regression, Kaplan-Meier plots, and Cox models. RESULTS: Making a CD4(+) T-cell ELISpot response (n = 119) at enrolment was associated with higher CD4(+) cell counts (P = 0.02) and to some extent lower plasma HIV RNA (P = 0.07). There was no correlation between the number of overlapping Gag CD8(+) T-cell ELISpot responses (n = 138) and plasma HIV-1 RNA viral load. Over a median follow-up of 2.9 years, baseline CD4(+) cell ELISpot responses (n = 119) were associated with slower clinical progression (P = 0.01; log-rank). Over a median of 3.1 years, there was no evidence for a survival advantage imposed by CD8(+) T-cell immunity (P = 0.82). CONCLUSION: These data support a dominant protective role for CD4(+) T-cell immunity in PHI compared with CD8(+) T-cell responses, and are highly pertinent to HIV pathogenesis and vaccines, indicating that vaccine-induced CD4(+) responses may confer sustained benefit. Hide abstract

Purcell DF, Elliott JH, Ross AL, Frater J. 2013. Towards an HIV cure: science and debate from the International AIDS Society 2013 symposium. Retrovirology, 10 (1), pp. 134. Read abstract | Read more

The International AIDS Society convened the multi-stakeholder "Towards an HIV Cure" symposium in Kuala Lumpur, Malaysia in 2013 to address the significant research challenges posed by the search for a cure for HIV infection. Current antiretroviral regimens select for a small reservoir of cells that harbour latent HIV provirus, produce few or no HIV virions, and resist detection or clearance by host immunity. The symposium examined basic molecular science and animal model data, and emerging and ongoing clinical trial results to prioritise strategies and determine the viral and immune responses that could lead to HIV remission without ART. Here we review the presentations that scrutinized the molecular mechanisms controlling virus expression from proviral DNA, and the intrinsic cellular restriction and immune mechanisms preventing viral production. Insights from the basic science have translated into new therapeutic strategies seeking HIV remission without ongoing therapy, and much interest was focused on these ongoing trials. We also summarise the emerging ethical issues and patient expectations as concepts move into the clinic. Hide abstract

Williams JP, Southern P, Lissina A, Christian HC, Sewell AK, Phillips R, Pankhurst Q, Frater J. 2013. Application of magnetic field hyperthermia and superparamagnetic iron oxide nanoparticles to HIV-1-specific T-cell cytotoxicity. Int J Nanomedicine, 8 pp. 2543-2554. Read abstract | Read more

The latent HIV-1 reservoir remains the major barrier to HIV-1 eradication. Although successful at limiting HIV replication, highly active antiretroviral therapy is unable to cure HIV infection, thus novel therapeutic strategies are needed to eliminate the virus. Magnetic field hyperthermia (MFH) generates thermoablative cytotoxic temperatures in target-cell populations, and has delivered promising outcomes in animal models, as well as in several cancer clinical trials. MFH has been proposed as a strategy to improve the killing of HIV-infected cells and for targeting the HIV latent reservoirs. We wished to determine whether MFH could be used to enhance cytotoxic T-lymphocyte (CTL) targeting of HIV-infected cells in a proof-of-concept study. Here, for the first time, we apply MFH to an infectious disease (HIV-1) using the superparamagnetic iron oxide nanoparticle FeraSpin R. We attempt to improve the cytotoxic potential of T-cell receptor-transfected HIV-specific CTLs using thermotherapy, and assess superparamagnetic iron oxide nanoparticle toxicity, uptake, and effect on cell function using more sensitive methods than previously described. FeraSpin R exhibited only limited toxicity, demonstrated efficient uptake and cell-surface attachment, and only modestly impacted T-cell function. In contrast to the cancer models, insufficient MFH was generated to enhance CTL killing of HIV-infected cells. MFH remains an exciting new technology in the field of cancer therapeutics, which, as technology improves, may have significant potential to enhance CTL function and act as an adjunctive therapy in the eradication of latently infected HIV-positive cells. Hide abstract

Palmer D, Frater J, Phillips R, McLean AR, McVean G. 2013. Integrating genealogical and dynamical modelling to infer escape and reversion rates in HIV epitopes. Proc Biol Sci, 280 (1762), pp. 20130696. Read abstract | Read more

The rates of escape and reversion in response to selection pressure arising from the host immune system, notably the cytotoxic T-lymphocyte (CTL) response, are key factors determining the evolution of HIV. Existing methods for estimating these parameters from cross-sectional population data using ordinary differential equations (ODEs) ignore information about the genealogy of sampled HIV sequences, which has the potential to cause systematic bias and overestimate certainty. Here, we describe an integrated approach, validated through extensive simulations, which combines genealogical inference and epidemiological modelling, to estimate rates of CTL escape and reversion in HIV epitopes. We show that there is substantial uncertainty about rates of viral escape and reversion from cross-sectional data, which arises from the inherent stochasticity in the evolutionary process. By application to empirical data, we find that point estimates of rates from a previously published ODE model and the integrated approach presented here are often similar, but can also differ several-fold depending on the structure of the genealogy. The model-based approach we apply provides a framework for the statistical analysis and hypothesis testing of escape and reversion in population data and highlights the need for longitudinal and denser cross-sectional sampling to enable accurate estimate of these key parameters. Hide abstract

James A, Blagojevic J, Benham SW, Cornall R, Frater J. 2013. Azathioprine hypersensitivity presenting as septic shock with encephalopathy. BMJ Case Rep, 2013 (mar18 1), pp. bcr2012008340-bcr2012008340. Read abstract | Read more

We present a case of azathioprine hypersensitivity presenting as septic shock with associated encephalopathy. The patient was presented with rapid onset of fever, hypotension, confusion and a rapidly declining conscious level. He was admitted to the intensive care unit where he received numerous invasive investigations and treatments with broad-spectrum antibiotics, antivirals and antifungals. All microbial cultures were negative. The patient-consistent with azathioprine hypersensitivity-made a spontaneous recovery after 7 days. The case shows that a time line of drug initiation is a key part of the medical history and consideration of azathioprine hypersensitivity could avoid unnecessary interventions and excessive antimicrobial use. Hide abstract

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. Read 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. Hide abstract

Yager N, Robinson N, Brown H, Flanagan P, Frater J, Fidler S, Weber J, Phillips R, SPARTAC Trial Investigators. 2013. Longitudinal analysis of an HLA-B*51-restricted epitope in integrase reveals immune escape in early HIV-1 infection. AIDS, 27 (3), pp. 313-323. Read abstract | Read more

OBJECTIVE: To fully define cytotoxic T-lymphocyte (CTL) escape variants of an HLA-B*51-restricted integrase epitope in early HIV-1 infection. DESIGN: Ninety-four longitudinally sampled acute/early HIV-1 subtype B-infected participants were assessed to determine HLA-B*51-restricted LPPVVAKEI (LI9) escape variants. METHODS: LI9 was sequenced at baseline and subsequent time points. Interferon-γ (IFNγ) ELISpot assays were performed using serial log dilutions of variant LI9 peptides to determine the cellular response and functional avidity. RESULTS: There is a significant association between HLA-B*51 expression and an evolving LI9 sequence from baseline to year 1 (P < 0.0001). We detected that the V32I and P30X polymorphisms emerged within HLA-B*51 participants over time. Reversion of the P30S polymorphism was observed by year 1 in one HLA-B*51 participant. LPPIIAKEI and LPSIVAKEI had significantly lower functional avidity compared with LPPVVAKEI and so may be less well recognized by LI9-specific CTLs; a positive IFNγ response to IPSVVAKEI was rarely seen. Functional avidity to wild-type LI9 inversely correlated with viral load (R = 0.448, P = 0.0485). CONCLUSION: Our results provide support for the role of HLA-B*51-restricted CTLs and functional avidity in the control of early HIV-1 infection. Hide abstract

SPARTAC Trial Investigators, Fidler S, Porter K, Ewings F, Frater J, Ramjee G, Cooper D, Rees H et al. 2013. Short-course antiretroviral therapy in primary HIV infection. N Engl J Med, 368 (3), pp. 207-217. Read abstract | Read more

BACKGROUND: Short-course antiretroviral therapy (ART) in primary human immunodeficiency virus (HIV) infection may delay disease progression but has not been adequately evaluated. METHODS: We randomly assigned adults with primary HIV infection to ART for 48 weeks, ART for 12 weeks, or no ART (standard of care), with treatment initiated within 6 months after seroconversion. The primary end point was a CD4+ count of less than 350 cells per cubic millimeter or long-term ART initiation. RESULTS: A total of 366 participants (60% men) underwent randomization to 48-week ART (123 participants), 12-week ART (120), or standard care (123), with an average follow-up of 4.2 years. The primary end point was reached in 50% of the 48-week ART group, as compared with 61% in each of the 12-week ART and standard-care groups. The average hazard ratio was 0.63 (95% confidence interval [CI], 0.45 to 0.90; P=0.01) for 48-week ART as compared with standard care and was 0.93 (95% CI, 0.67 to 1.29; P=0.67) for 12-week ART as compared with standard care. The proportion of participants who had a CD4+ count of less than 350 cells per cubic millimeter was 28% in the 48-week ART group, 40% in the 12-week group, and 40% in the standard-care group. Corresponding values for long-term ART initiation were 22%, 21%, and 22%. The median time to the primary end point was 65 weeks (95% CI, 17 to 114) longer with 48-week ART than with standard care. Post hoc analysis identified a trend toward a greater interval between ART initiation and the primary end point the closer that ART was initiated to estimated seroconversion (P=0.09), and 48-week ART conferred a reduction in the HIV RNA level of 0.44 log(10) copies per milliliter (95% CI, 0.25 to 0.64) 36 weeks after the completion of short-course therapy. There were no significant between-group differences in the incidence of the acquired immunodeficiency syndrome, death, or serious adverse events. CONCLUSIONS: A 48-week course of ART in patients with primary HIV infection delayed disease progression, although not significantly longer than the duration of the treatment. There was no evidence of adverse effects of ART interruption on the clinical outcome. (Funded by the Wellcome Trust; SPARTAC number, ISRCTN76742797, and EudraCT number, 2004-000446-20.). Hide abstract

Kløverpris HN, Harndahl M, Leslie AJ, Carlson JM, Ismail N, van der Stok M, Huang KH, Chen F et al. 2012. HIV control through a single nucleotide on the HLA-B locus. J Virol, 86 (21), pp. 11493-11500. Read abstract | Read more

Genetic variation within the HLA-B locus has the strongest impact on HIV disease progression of any polymorphisms within the human genome. However, identifying the exact mechanism involved is complicated by several factors. HLA-Bw4 alleles provide ligands for NK cells and for CD8 T cells, and strong linkage disequilibrium between HLA class I alleles complicates the discrimination of individual HLA allelic effects from those of other HLA and non-HLA alleles on the same haplotype. Here, we exploit an experiment of nature involving two recently diverged HLA alleles, HLA-B*42:01 and HLA-B*42:02, which differ by only a single amino acid. Crucially, they occur primarily on identical HLA class I haplotypes and, as Bw6 alleles, do not act as NK cell ligands and are therefore largely unconfounded by other genetic factors. We show that in an outbred cohort (n = 2,093) of HIV C-clade-infected individuals, a single amino acid change at position 9 of the HLA-B molecule critically affects peptide binding and significantly alters the cytotoxic T lymphocyte (CTL) epitopes targeted, measured directly ex vivo by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay (P = 2 × 10(-10)) and functionally through CTL escape mutation (P = 2 × 10(-8)). HLA-B*42:01, which presents multiple Gag epitopes, is associated with a 0.52 log(10) lower viral-load set point than HLA-B*42:02 (P = 0.02), which presents no p24 Gag epitopes. The magnitude of this effect from a single amino acid difference in the HLA-A*30:01/B*42/Cw*17:01 haplotype is equivalent to 75% of that of HLA-B*57:03, the most protective HLA class I allele in this population. This naturally controlled experiment represents perhaps the clearest demonstration of the direct impact of a particular HIV-specific CTL on disease control. Hide abstract

Matthews PC, Listgarten J, Carlson JM, Payne R, Huang KH, Frater J, Goedhals D, Steyn D et al. 2012. Co-operative additive effects between HLA alleles in control of HIV-1. PLoS One, 7 (10), pp. e47799. Read abstract | Read more

BACKGROUND: HLA class I genotype is a major determinant of the outcome of HIV infection, and the impact of certain alleles on HIV disease outcome is well studied. Recent studies have demonstrated that certain HLA class I alleles that are in linkage disequilibrium, such as HLA-A*74 and HLA-B*57, appear to function co-operatively to result in greater immune control of HIV than mediated by either single allele alone. We here investigate the extent to which HLA alleles--irrespective of linkage disequilibrium--function co-operatively. METHODOLOGY/PRINCIPAL FINDINGS: We here refined a computational approach to the analysis of >2000 subjects infected with C-clade HIV first to discern the individual effect of each allele on disease control, and second to identify pairs of alleles that mediate 'co-operative additive' effects, either to improve disease suppression or to contribute to immunological failure. We identified six pairs of HLA class I alleles that have a co-operative additive effect in mediating HIV disease control and four hazardous pairs of alleles that, occurring together, are predictive of worse disease outcomes (q<0.05 in each case). We developed a novel 'sharing score' to quantify the breadth of CD8+ T cell responses made by pairs of HLA alleles across the HIV proteome, and used this to demonstrate that successful viraemic suppression correlates with breadth of unique CD8+ T cell responses (p = 0.03). CONCLUSIONS/SIGNIFICANCE: These results identify co-operative effects between HLA Class I alleles in the control of HIV-1 in an extended Southern African cohort, and underline complementarity and breadth of the CD8+ T cell targeting as one potential mechanism for this effect. Hide abstract

Draenert R, Frater J, Prado JG. 2012. Virus Immune Evasion: New Mechanism and Implications in Disease Outcome Advances in Virology, 2012 pp. 1-1. | Read more

Fryer HR, Frater J, Duda A, Palmer D, Phillips RE, McLean AR. 2012. Cytotoxic T-lymphocyte escape mutations identified by HLA association favor those which escape and revert rapidly. J Virol, 86 (16), pp. 8568-8580. Read abstract | Read more

Identifying human immunodeficiency virus (HIV) immune escape mutations has implications for understanding the impact of host immunity on pathogen evolution and guiding the choice of vaccine antigens. One means of identifying cytotoxic-T-lymphocyte (CTL) escape mutations is to search for statistical associations between mutations and host human leukocyte antigen (HLA) class I alleles at the population level. The impact of evolutionary rates on the strength of such associations is not well defined. Here, we address this topic using a mathematical model of within-host evolution and between-host transmission of CTL escape mutants that predicts the prevalence of escape mutants at the population level. We ask how the rates at which an escape mutation emerges in a host who bears the restricting HLA and reverts when transmitted to a host who does not bear the HLA affect the strength of an association. We consider the impact of these factors when using a standard statistical method to test for an association and when using an adaptation of that method that corrects for phylogenetic relationships. We show that with both methods, the average sample size required to identify an escape mutation is smaller if the mutation escapes and reverts quickly. Thus, escape mutations identified as HLA associated systematically favor those that escape and revert rapidly. We also present expressions that can be used to infer escape and reversion rates from cross-sectional escape prevalence data. Hide abstract

Carlson JM, Listgarten J, Pfeifer N, Tan V, Kadie C, Walker BD, Ndung'u T, Shapiro R et al. 2012. Widespread impact of HLA restriction on immune control and escape pathways of HIV-1. J Virol, 86 (9), pp. 5230-5243. Read abstract | Read more

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine. Hide abstract

Hamlyn E, Hickling S, Porter K, Frater J, Phillips R, Robinson M, Mackie NE, Kaye S, McClure M, Fidler S, SPARTAC Investigators. 2012. Increased levels of CD4 T-cell activation in individuals with CXCR4 using viruses in primary HIV-1 infection. AIDS, 26 (7), pp. 887-890. Read abstract | Read more

CXCR4-tropic (X4) HIV-1 variants are associated with faster disease progression compared with CCR5-tropic variants; however, the mechanism for this is unclear. We measured T-cell activation in 120 individuals with primary HIV-1 infection. X4-utilizing variants, determined genotypically, were present in 8.3% of the participants and were associated with higher levels of CD4 T-cell activation, even after adjusting for other prognostic factors. Increased CD4 T-cell activation may influence the more rapid immunological decline associated with X4 virus. Hide abstract

Williams JP, Frater J. 2012. Current understanding in HIV immunopathology and treatment. QJM, 105 (8), pp. 725-728. | Read more

Frater J. 2011. New approaches in HIV eradication research. Curr Opin Infect Dis, 24 (6), pp. 593-598. Read abstract | Read more

PURPOSE OF REVIEW: Despite the proven efficacy of highly active antiretroviral therapy in reducing mortality and morbidity of HIV infection, longer-term strategies are less well defined and there is renewed interest in HIV eradication. This review will describe the major obstacles that need to be overcome and the key new advances and strategies designed to achieve an HIV cure. RECENT FINDINGS: Characterization of the HIV viral reservoir over the past few years has led to a better understanding of which approaches might successfully lead to eradication. A number of approaches such as histone modification, immunotoxins, gene therapy and gene knockout strategies have resulted and have been explored initially in vitro. There has been progression from both laboratory and animal model studies, and clinical trials are now underway using new approaches such as histone deacetylase inhibitors and zinc finger nucleases. SUMMARY: Although there is currently no cure for HIV infection, there has been a resurgence of interest in the field with the development of a number of potential new approaches, some of which have entered clinical trials. Hide abstract

English S, Katzourakis A, Bonsall D, Flanagan P, Duda A, Fidler S, Weber J, McClure M, SPARTAC Trial Investigators, Phillips R, Frater J. 2011. Phylogenetic analysis consistent with a clinical history of sexual transmission of HIV-1 from a single donor reveals transmission of highly distinct variants. Retrovirology, 8 (1), pp. 54. Read abstract | Read more

BACKGROUND: To combat the pandemic of human immunodeficiency virus 1 (HIV-1), a successful vaccine will need to cope with the variability of transmissible viruses. Human hosts infected with HIV-1 potentially harbour many viral variants but very little is known about viruses that are likely to be transmitted, or even if there are viral characteristics that predict enhanced transmission in vivo. We show for the first time that genetic divergence consistent with a single transmission event in vivo can represent several years of pre-transmission evolution. RESULTS: We describe a highly unusual case consistent with a single donor transmitting highly related but distinct HIV-1 variants to two individuals on the same evening. We confirm that the clustering of viral genetic sequences, present within each recipient, is consistent with the history of a single donor across the viral env, gag and pol genes by maximum likelihood and bayesian Markov Chain Monte Carlo based phylogenetic analyses. Based on an uncorrelated, lognormal relaxed clock of env gene evolution calibrated with other datasets, the time since the most recent common ancestor is estimated as 2.86 years prior to transmission (95% confidence interval 1.28 to 4.54 years). CONCLUSION: Our results show that an effective design for a preventative vaccine will need to anticipate extensive HIV-1 diversity within an individual donor as well as diversity at the population level. Hide abstract

Matthews PC, Adland E, Listgarten J, Leslie A, Mkhwanazi N, Carlson JM, Harndahl M, Stryhn A et al. 2011. HLA-A*7401-mediated control of HIV viremia is independent of its linkage disequilibrium with HLA-B*5703. J Immunol, 186 (10), pp. 5675-5686. Read abstract | Read more

The potential contribution of HLA-A alleles to viremic control in chronic HIV type 1 (HIV-1) infection has been relatively understudied compared with HLA-B. In these studies, we show that HLA-A*7401 is associated with favorable viremic control in extended southern African cohorts of >2100 C-clade-infected subjects. We present evidence that HLA-A*7401 operates an effect that is independent of HLA-B*5703, with which it is in linkage disequilibrium in some populations, to mediate lowered viremia. We describe a novel statistical approach to detecting additive effects between class I alleles in control of HIV-1 disease, highlighting improved viremic control in subjects with HLA-A*7401 combined with HLA-B*57. In common with HLA-B alleles that are associated with effective control of viremia, HLA-A*7401 presents highly targeted epitopes in several proteins, including Gag, Pol, Rev, and Nef, of which the Gag epitopes appear immunodominant. We identify eight novel putative HLA-A*7401-restricted epitopes, of which three have been defined to the optimal epitope. In common with HLA-B alleles linked with slow progression, viremic control through an HLA-A*7401-restricted response appears to be associated with the selection of escape mutants within Gag epitopes that reduce viral replicative capacity. These studies highlight the potentially important contribution of an HLA-A allele to immune control of HIV infection, which may have been concealed by a stronger effect mediated by an HLA-B allele with which it is in linkage disequilibrium. In addition, these studies identify a factor contributing to different HIV disease outcomes in individuals expressing HLA-B*5703. Hide abstract

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. Read 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. Hide abstract

Barnes E, Flanagan P, Brown A, Robinson N, Brown H, McClure M, Oxenius A, Collier J et al. 2010. Failure to detect xenotropic murine leukemia virus-related virus in blood of individuals at high risk of blood-borne viral infections. J Infect Dis, 202 (10), pp. 1482-1485. Read abstract | Read more

A xenotropic murine leukemia virus-related virus (XMRV) has recently been reported in association with prostate cancer and chronic fatigue syndrome, with a prevalence of up to 3.7% in the healthy population. We looked for XMRV in 230 patients with human immunodeficiency virus type 1 or hepatitis C infection. XMRV was undetectable in plasma or peripheral blood mononuclear cells by polymerase chain reaction targeting XMRV gag or env. T cell responses to XMRV Gag were undetectable in peripheral blood mononuclear cells by ex vivo gamma interferon enzyme-linked immunospot assay. In our cohorts, XMRV was not enriched in patients with blood-borne or sexually transmitted infections from the United Kingdom and Western Europe. Hide abstract

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. Read 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. Hide abstract

Hamlyn E, Hickling S, Frater J, Phillips R, Babiker A, McClure M, Fidler S. 2010. Protective HLA class I alleles are associated with reduced immune activation and fibrinolysis in individuals with primary HIV infection HIV MEDICINE, 11 pp. 3-3.

Fryer HR, Frater J, Duda A, Roberts MG, SPARTAC Trial Investigators, Phillips RE, McLean AR. 2010. Modelling the evolution and spread of HIV immune escape mutants. PLoS Pathog, 6 (11), pp. e1001196. Read abstract | Read more

During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level. Hide abstract

Matthews P, Prendergast A, Kawashima Y, Pfafferott K, Frater J, Heckerman D, Takiguchi M, Goulder P. 2009. INTER-CONTINENTAL PATTERNS OF HIV-1 CONTROL: HLA AND THE QUEST FOR A T CELL VACCINE JOURNAL OF INFECTION, 59 (6), pp. S430-S431.

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. Read 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. Hide abstract

English S, Katzourakis A, Flanagan P, Duda A, Francis J, Matthews P, Prendergast A, Goulder P et al. 2009. Contemporaneous transmission of genetically distinct HIV variants from a single donor to two recipients RETROVIROLOGY, 6

Duda A, Lee-Turner L, Fox J, Robinson N, Dustan S, Kaye S, Fryer H, Carrington M et al. 2009. HLA-associated clinical progression correlates with epitope reversion rates in early human immunodeficiency virus infection. J Virol, 83 (3), pp. 1228-1239. Read abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) can evade immunity shortly after transmission to a new host but the clinical significance of this early viral adaptation in HIV infection is not clear. We present an analysis of sequence variation from a longitudinal cohort study of HIV adaptation in 189 acute seroconverters followed for up to 3 years. We measured the rates of variation within well-defined epitopes to determine associations with the HLA-linked hazard of disease progression. We found early reversion across both the gag and pol genes, with a 10-fold faster rate of escape in gag (2.2 versus 0.27 forward mutations/1,000 amino acid sites). For most epitopes (23/34), variation in the HLA-matched and HLA-unmatched controls was similar. For a minority of epitopes (8/34, and generally associated with HLA class I alleles that confer clinical benefit), new variants appeared early and consistently over the first 3 years of infection. Reversion occurred early at a rate which was HLA-dependent and correlated with the HLA class 1-associated relative hazard of disease progression and death (P = 0.0008), reinforcing the association between strong cytotoxic T-lymphocyte responses, viral fitness, and disease status. These data provide a comprehensive overview of viral adaptation in the first 3 years of infection. Our findings of HLA-dependent reversion suggest that costs are borne by some escape variants which may benefit the host, a finding contrary to a simple immune evasion paradigm. These epitopes, which are both strongly and frequently recognized, and for which escape involves a high cost to the virus, have the potential to optimize vaccine design. Hide abstract

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. Read 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. Hide abstract

Frater AJ, Brown H, Oxenius A, Günthard HF, Hirschel B, Robinson N, Leslie AJ, Payne R et al. 2007. Effective T-cell responses select human immunodeficiency virus mutants and slow disease progression. J Virol, 81 (12), pp. 6742-6751. Read abstract | Read more

The possession of some HLA class I molecules is associated with delayed progression to AIDS. The mechanism behind this beneficial effect is unclear. We tested the idea that cytotoxic T-cell responses restricted by advantageous HLA class I molecules impose stronger selection pressures than those restricted by other HLA class I alleles. As a measure of the selection pressure imposed by HLA class I alleles, we determined the extent of HLA class I-associated epitope variation in a cohort of European human immunodeficiency virus (HIV)-positive individuals (n=84). We validated our findings in a second, distinct cohort of African patients (n=516). We found that key HIV epitopes restricted by advantageous HLA molecules (B27, B57, and B51 in European patients and B5703, B5801, and B8101 in African patients) were more frequently mutated in individuals bearing the restricting HLA than in those who lacked the restricting HLA class I molecule. HLA alleles associated with clinical benefit restricted certain epitopes for which the consensus peptides were frequently recognized by the immune response despite the circulating virus's being highly polymorphic. We found a significant inverse correlation between the HLA-associated hazard of disease progression and the mean HLA-associated prevalence of mutations within epitopes (P=0.028; R2=0.34). We conclude that beneficial HLA class I alleles impose strong selection at key epitopes. This is revealed by the frequent association between effective T-cell responses and circulating viral escape mutants and the rarity of these variants in patients who lack these favorable HLA class I molecules, suggesting a significant pressure to revert. Hide abstract

Zimbwa P, Milicic A, Frater J, Scriba TJ, Willis A, Goulder PJ, Pillay T, Gunthard H, Weber JN, Zhang HT, Phillips RE. 2007. Precise identification of a human immunodeficiency virus type 1 antigen processing mutant. J Virol, 81 (4), pp. 2031-2038. Read abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences. Hide abstract

Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, Reddy S, de Pierres C et al. 2007. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med, 13 (1), pp. 46-53. Read abstract | Read more

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation. Hide abstract

Frater AJ, Edwards CT, McCarthy N, Fox J, Brown H, Milicic A, Mackie N, Pillay T et al. 2006. Passive sexual transmission of human immunodeficiency virus type 1 variants and adaptation in new hosts. J Virol, 80 (14), pp. 7226-7234. Read abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences. Hide abstract

Scherer A, Frater J, Oxenius A, Agudelo J, Price DA, Günthard HF, Barnardo M, Perrin L et al. 2004. Quantifiable cytotoxic T lymphocyte responses and HLA-related risk of progression to AIDS. Proc Natl Acad Sci U S A, 101 (33), pp. 12266-12270. Read abstract | Read more

There are significant associations between possession of certain HLA class I alleles and rate of progression to AIDS. Immunological data provide an explanatory mechanism for this relationship. Patients with HLA types associated with rapid disease progression recognize a significantly smaller fraction of their known repertoire of viral epitopes than do patients with HLA types associated with slow progression. Population frequency of HLA types (or supertypes) and their capacity to elicit cytotoxic T lymphocyte responses are also negatively correlated. These data provide an immunological mechanism to explain HLA-related risk of progression to AIDS and emphasize the central role of viral evolution in the pathogenesis of HIV. Hide abstract

Beddows S, Galpin S, Kazmi SH, Ashraf A, Johargy A, Frater AJ, White N, Braganza R, Clarke J, McClure M, Weber JN. 2003. Performance of two commercially available sequence-based HIV-1 genotyping systems for the detection of drug resistance against HIV type 1 group M subtypes. J Med Virol, 70 (3), pp. 337-342. Read abstract | Read more

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved. Hide abstract

Myint L, Ariyoshi K, Yan H, Frater AJ, Auwanit W, Pathipvanith P, Yamada K, Matsuda M et al. 2002. Mutagenically separated PCR assay for rapid detection of M41L and K70R zidovudine resistance mutations in CRF01_AE (subtype E) human immunodeficiency virus type 1. Antimicrob Agents Chemother, 46 (12), pp. 3861-3868. Read abstract | Read more

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries. Hide abstract

Frater J. 2002. The impact of HIV-1 subtype on the clinical response on HAART. J HIV Ther, 7 (4), pp. 92-96. Read abstract

Highly active antiretroviral therapy (HAART) has led to a reduction in HIV-related mortality and morbidity. Most patients who have benefited from HAART are infected with HIV-1 subtype B, which predominates in Western Europe, the USA and the rest of the industrialised world. However, most HIV-infected people live in sub-Saharan Africa, Asia and Eastern Europe. In these areas, subtypes other than B are responsible for the epidemic of HIV-1 infection. This review focuses on the clinical significance of HIV-1 infection with a non-B subtype. The increase in availability of HAART to developing countries together with the large number of HIV-1-infected immigrants being treated in the industrialised world means that data on the clinical response to therapy for non-B HIV-1 infections are becoming of greater practical relevance. If antiretroviral agents, which generally target subtype B, are less efficacious in non-B infections, this will have major clinical implications for therapeutic strategies. Data on drug susceptibility, clinical response and the development of resistance in non-B HIV-1 subtypes are discussed here. Hide abstract

Frater AJ, Dunn DT, Beardall AJ, Ariyoshi K, Clarke JR, McClure MO, Weber JN. 2002. Comparative response of African HIV-1-infected individuals to highly active antiretroviral therapy. AIDS, 16 (8), pp. 1139-1146. Read abstract | Read more

OBJECTIVE: Few data exist on the virological response to antiretroviral therapy of individuals infected with African HIV-1 subtypes. Our objective was to compare the response, in our clinic, of African HIV-1-infected patients with their British and European contemporaries treated with the same regimes. DESIGN: The St Mary's Hospital HIV database was used to identify drug-naive African and European patients starting a highly active antiretroviral therapy (HAART) regimen. METHODS: HIV-1 subtype was determined by phylogenetic analysis of pol sequences. Kaplan-Meier survival analysis was used to estimate the proportion of patients achieving undetectable viral loads (< 500 copies/ml). The longer-term response to therapy was assessed by changes in CD4 cell counts and viral loads from baseline. RESULTS: A total of 265 patients were classified as 'European' and 97 as 'African', confirmed by sequence. The time to first undetectable viral load was similar for the two groups (P = 0.9). Although there were no statistically significant differences in the CD4 cell count responses (P = 0.11), there was evidence of an increase in viral load after 9 months for the African group, resulting in a widening viral load gap between the two cohorts; the effect of ethnic group was statistically significant (P < 0.001). CONCLUSION: The initial virological and immunological responses of the African and European cohorts to HAART were similar; although the longer-term virological response was poorer in the African cohort, which may be related to adherence. On the basis of these findings, there is no justification for withholding HAART from Africa on virological grounds. Hide abstract

Frater J, Dunn D, Weber JN, McClure MO. 2002. Association between secondary mutations in human immunodeficiency virus type 1 protease and therapeutic outcome. J Infect Dis, 185 (9), pp. 1376. | Read more

Fidler S, Frater J, Clarke J, Weber J. 2001. HIV-1 drug resistance in primary infections in the UK. BMJ, 323 (7313), pp. 632-633. | Read more

Frater AJ, Beardall A, Ariyoshi K, Churchill D, Galpin S, Clarke JR, Weber JN, McClure MO. 2001. Impact of baseline polymorphisms in RT and protease on outcome of highly active antiretroviral therapy in HIV-1-infected African patients. AIDS, 15 (12), pp. 1493-1502. Read abstract | Read more

OBJECTIVE: To assess the therapeutic response and investigate the significance of polymorphic codons in African patients receiving highly-active antiretroviral therapy (HAART). DESIGN AND METHODS: African patients were identified from the St Mary's Hospital HIV-1 database. Clinical outcome was assessed by viral load and CD4 cell count. Pre- and post-therapy sequences of RT and protease were analysed. The impact of subtype and individual polymorphic codons on therapeutic outcome was assessed statistically (Fishers exact and chi2 tests) and phylogenetically (Jukes and Cantor). RESULTS: Of 79 drug-naive African patients who were prescribed HAART, 60 remained undetectable for 1 year, with no differences detected in the clinical response to non-nucleoside reverse transcriptase inhibitor (NNRTI)- or protease inhibitor (PI)-containing regimes. Country of origin, sex and viral subtype had no impact on outcome of HAART. A total of 133 polymorphisms were identified in pol (37 in protease and 96 in RT), with a mean of 9.0 in protease and 22.3 in RT per patient. There was no significant difference in the overall numbers of polymorphisms per patient, and no single polymorphism had any impact on clinical outcome. Sequences from 'failing' patients experiencing viral rebound produced few mutations known to be associated with drug resistance, suggesting minimal drug pressure. CONCLUSIONS: The response of patients infected with African subtypes of HIV-1 to HAART appears to be independent of regime, HIV-1 clade and baseline polymorphisms. Non-B subtypes are fully sensitive to HAART and, accordingly, therapy should not be withheld from African patients for reasons of viral diversity. Hide abstract

Frater AJ, Chaput CC, Beddows S, Weber JN, McClure MO. 2001. Simple detection of point mutations associated with HIV-1 drug resistance. J Virol Methods, 93 (1-2), pp. 145-156. Read abstract | Read more

A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS--PCR) --- a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant--wild-type population. Gene sequencing was carried out simultaneously for comparison. MS--PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS--PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS--PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS--PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest. Hide abstract

Rosenblum LL, Patton G, Grigg AR, Frater AJ, Cain D, Erlwein O, Hill CL, Clarke JR, McClure MO. 2001. Differential susceptibility of retroviruses to nucleoside analogues. Antivir Chem Chemother, 12 (2), pp. 91-97. Read abstract | Read more

Retroviruses may cause diseases in their vertebrate hosts. They are distinguished by their common means of replication involving reverse transcription, a process inhibited by nucleoside reverse transcriptase inhibitors (NRTIs) and other compounds used in antiretroviral chemotherapy. Previous work on NRTIs has been limited to their effect on human immunodeficiency virus (HIV) (for review see Ho & Hitchcock, 1989; Weller, 1999) and little information exists regarding the efficacy and therapeutic potential of these drugs against other retroviruses. We have tested all six NRTIs licensed for HIV treatment [didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), zidovudine (AZT) and abacavir (ABC)] against seven retroviruses representative of the traditional subfamilies: Spumavirinae, Lentivirinae and the Oncovirinae. As expected, each drug showed a range of activities against the panel of retroviruses, some drugs inhibiting other viruses at concentrations well below those required for HIV. Overall, AZT was the most active inhibitor (IC50 range, 0.032-1.0 microM), being most active against the Spuma (foamy) viruses. Abacavir was inhibitory for HIV-1, MN strain (HIV-1 MN), amphotrophic murine leukemia virus (MLV-A) and simian foamy virus type 6 (SFV-6). The least effective inhibitor, 3TC (IC50 range, 0.32->100 microM), was most potent against simian retrovirus types 1 and 2 (SRV-1, SRV-2) and HIV-1, but did not inhibit foamy viruses and MLV-A. Additionally, there were differences in the concentration of drug required to inhibit closely related viruses. Taken together, these data suggest that NRTIs have a wide spectrum of antiretroviral activity and the activity of compounds, even against closely related retroviruses, cannot be predicted. Hide abstract

Frater AJ, Galpin S, Beardall A, Clarke JR, Ariyoshi K, Weber JN, McClure MO. 2000. Baseline polymorphisms found in patients infected with non-clade BHIV-1 do not affect outcome to HAART AIDS, 14 pp. S114-S114.

Frater AJ, Chaput CC, Weber JN, McClure MO. 2000. HIV-1 resistance genotyping by sequencing produces inconsistent results for mixed viral populations. AIDS, 14 (10), pp. 1473-1475. | Read more

Next Generation Sequencing Approaches to the HIV-1 Reservoir

There is no cure for HIV infection due to the persistence of HIV in a latent form in a reservoir of resting T lymphocytes. Although around 1 in 1000 CD4 T cells may contain integrated HIV DNA, the vast majority of this is replication incompetent. NGS has the potential for applying unbiased PCR-free approaches to sequence the HIV reservoir, and provide vital information linking viral variation to clinical phenotype and immunological responses. Application of bioinformatic NGS tools will overcome ...

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