register interest

Professor Sir Andrew J McMichael

Research Area: Immunology
Scientific Themes: Immunology & Infectious Disease
Keywords: HIV, Vaccines, T cell immunity and Influenza
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Our HIV vaccines have completed five small phase I clinical trials and one large phase I/II clinical trials in London and Oxford. The vaccine is DNA encoding HIV clade A gag p24 and p17 plus a string of epitopes, and the same inserted into recombinant Modified Vaccinia Virus Ankara (MVA). Both stimulate strong CD8+ T cell responses in mice and macaques. In humans both have stimulated measurable CD8+ T cell responses in HIV low risk, uninfected volunteers. Current work is focussing on improving immunogenicity and working out assays for measuring immune responses that are likely to be protective against HIV infection. We tested the effect of those vaccines on boosting T cell responses in HIV infected patients who are on anti-retroviral drugs, with the aim of interrupting drug treatment when the T cell response is enhanced. Encouraging boosts are seen using the MVA-HIVA vaccine.
Nef (key virus protein in virus pathogenesis) initiates a programme of transcription closely similar to that triggered by T cell receptor activation. This favours HIV virus replication. Nef also alters the composition of proteins in cell signalling site, and exclusion of certain regulators favours the activation state.
We continue to explore selection of HIV-1 mutants by the immune response. T cells are very sensitive to small changes in the composition, orientation or flexibility of the exposed peptide bound to HLA. We are studying the immunodominant SLYNTVATL epitope presented by HLA-A2, showing selection of a sequence of mutations that impair T cell recognition. Why the T cells do not respond by new primary responses to the variants is also under investigation.

Name Department Institution Country
Professor Barton Haynes Duke University, North Carolina United States
Haynes BF, Shaw GM, Korber B, Kelsoe G, Sodroski J, Hahn BH, Borrow P, McMichael AJ. 2016. HIV-Host Interactions: Implications for Vaccine Design. Cell Host Microbe, 19 (3), pp. 292-303. | Show Abstract | Read more

Development of an effective AIDS vaccine is a global priority. However, the extreme diversity of HIV type 1 (HIV-1), which is a consequence of its propensity to mutate to escape immune responses, along with host factors that prevent the elicitation of protective immune responses, continue to hinder vaccine development. Breakthroughs in understanding of the biology of the transmitted virus, the structure and nature of its envelope trimer, vaccine-induced CD8 T cell control in primates, and host control of broadly neutralizing antibody elicitation have given rise to new vaccine strategies. Despite this promise, emerging data from preclinical trials reinforce the need for additional insight into virus-host biology in order to facilitate the development of a successful vaccine.

Hansen SG, Wu HL, Burwitz BJ, Hughes CM, Hammond KB, Ventura AB, Reed JS, Gilbride RM et al. 2016. Broadly targeted CD8⁺ T cell responses restricted by major histocompatibility complex E. Science, 351 (6274), pp. 714-720. | Show Abstract | Read more

Major histocompatibility complex E (MHC-E) is a highly conserved, ubiquitously expressed, nonclassical MHC class Ib molecule with limited polymorphism that is primarily involved in the regulation of natural killer (NK) cells. We found that vaccinating rhesus macaques with rhesus cytomegalovirus vectors in which genes Rh157.5 and Rh157.4 are deleted results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8αβ(+) T cells, at ~4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side-chain interactions within a stable, open binding groove. Because MHC-E is up-regulated to evade NK cell activity in cells infected with HIV, simian immunodeficiency virus, and other persistent viruses, MHC-E-restricted CD8(+) T cell responses have the potential to exploit pathogen immune-evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.

Ondondo B, Murakoshi H, Clutton G, Abdul-Jawad S, Wee EG, Gatanaga H, Oka S, McMichael AJ, Takiguchi M, Korber B, Hanke T. 2016. Novel Conserved-region T-cell Mosaic Vaccine With High Global HIV-1 Coverage Is Recognized by Protective Responses in Untreated Infection. Mol Ther, 24 (4), pp. 832-842. | Show Abstract | Read more

An effective human immunodeficiency virus type 1 (HIV-1) vaccine is the best solution for halting the acquired immune deficiency syndrome epidemic. Here, we describe the design and preclinical immunogenicity of T-cell vaccine expressing novel immunogens tHIVconsvX, vectored by DNA, simian (chimpanzee) adenovirus, and poxvirus modified vaccinia virus Ankara (MVA), a combination highly immunogenic in humans. The tHIVconsvX immunogens combine the three leading strategies for elicitation of effective CD8(+) T cells: use of regions of HIV-1 proteins functionally conserved across all M group viruses (to make HIV-1 escape costly on viral fitness), inclusion of bivalent complementary mosaic immunogens (to maximize global epitope matching and breadth of responses, and block common escape paths), and inclusion of epitopes known to be associated with low viral load in infected untreated people (to induce field-proven protective responses). tHIVconsvX was highly immunogenic in two strains of mice. Furthermore, the magnitude and breadth of CD8(+) T-cell responses to tHIVconsvX-derived peptides in treatment-naive HIV-1(+) patients significantly correlated with high CD4(+) T-cell count and low viral load. Overall, the tHIVconsvX design, combining the mosaic and conserved-region approaches, provides an indisputably better coverage of global HIV-1 variants than previous T-cell vaccines. These immunogens delivered in a highly immunogenic framework of adenovirus prime and MVA boost are ready for clinical development.

Barton JP, Goonetilleke N, Butler TC, Walker BD, McMichael AJ, Chakraborty AK. 2016. Relative rate and location of intra-host HIV evolution to evade cellular immunity are predictable. Nat Commun, 7 pp. 11660. | Show Abstract | Read more

Human immunodeficiency virus (HIV) evolves within infected persons to escape being destroyed by the host immune system, thereby preventing effective immune control of infection. Here, we combine methods from evolutionary dynamics and statistical physics to simulate in vivo HIV sequence evolution, predicting the relative rate of escape and the location of escape mutations in response to T-cell-mediated immune pressure in a cohort of 17 persons with acute HIV infection. Predicted and clinically observed times to escape immune responses agree well, and we show that the mutational pathways to escape depend on the viral sequence background due to epistatic interactions. The ability to predict escape pathways and the duration over which control is maintained by specific immune responses open the door to rational design of immunotherapeutic strategies that might enable long-term control of HIV infection. Our approach enables intra-host evolution of a human pathogen to be predicted in a probabilistic framework.

Jallow S, Leligdowicz A, Kramer HB, Onyango C, Cotten M, Wright C, Whittle HC, McMichael A, Dong T, Kessler BM, Rowland-Jones SL. 2015. Correction. The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated. Eur J Immunol, 45 (9), pp. 2701. | Read more

Jallow S, Leligdowicz A, Kramer HB, Onyango C, Cotten M, Wright C, Whittle HC, McMichael A, Dong T, Kessler BM, Rowland-Jones SL. 2015. The presence of prolines in the flanking region of an immunodominant HIV-2 gag epitope influences the quality and quantity of the epitope generated. Eur J Immunol, 45 (8), pp. 2232-2242. | Show Abstract | Read more

Both the recognition of HIV-infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV-2 gag-specific T-cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165 DRFYKSLRA173 , within the highly conserved Major Homology Region of gag-p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag-p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag-specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165 DRFYKSLRA173 epitope. Our data demonstrate that the 165 DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral-load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV-2 encoding the "PPP-gag" sequence and both strong gag-specific CTL responses as well as lower viral load.

Hayward AC, Wang L, Goonetilleke N, Fragaszy EB, Bermingham A, Copas A, Dukes O, Millett ER et al. 2015. Natural T Cell-mediated Protection against Seasonal and Pandemic Influenza. Results of the Flu Watch Cohort Study. Am J Respir Crit Care Med, 191 (12), pp. 1422-1431. | Show Abstract | Read more

RATIONALE: A high proportion of influenza infections are asymptomatic. Animal and human challenge studies and observational studies suggest T cells protect against disease among those infected, but the impact of T-cell immunity at the population level is unknown. OBJECTIVES: To investigate whether naturally preexisting T-cell responses targeting highly conserved internal influenza proteins could provide cross-protective immunity against pandemic and seasonal influenza. METHODS: We quantified influenza A(H3N2) virus-specific T cells in a population cohort during seasonal and pandemic periods between 2006 and 2010. Follow-up included paired serology, symptom reporting, and polymerase chain reaction (PCR) investigation of symptomatic cases. MEASUREMENTS AND MAIN RESULTS: A total of 1,414 unvaccinated individuals had baseline T-cell measurements (1,703 participant observation sets). T-cell responses to A(H3N2) virus nucleoprotein (NP) dominated and strongly cross-reacted with A(H1N1)pdm09 NP (P < 0.001) in participants lacking antibody to A(H1N1)pdm09. Comparison of paired preseason and post-season sera (1,431 sets) showed 205 (14%) had evidence of infection based on fourfold influenza antibody titer rises. The presence of NP-specific T cells before exposure to virus correlated with less symptomatic, PCR-positive influenza A (overall adjusted odds ratio, 0.27; 95% confidence interval, 0.11-0.68; P = 0.005, during pandemic [P = 0.047] and seasonal [P = 0.049] periods). Protection was independent of baseline antibodies. Influenza-specific T-cell responses were detected in 43%, indicating a substantial population impact. CONCLUSIONS: Naturally occurring cross-protective T-cell immunity protects against symptomatic PCR-confirmed disease in those with evidence of infection and helps to explain why many infections do not cause symptoms. Vaccines stimulating T cells may provide important cross-protective immunity.

Smith NM, Mlcochova P, Watters SA, Aasa-Chapman MM, Rabin N, Moore S, Edwards SG, Garson JA et al. 2015. Proof-of-Principle for Immune Control of Global HIV-1 Reactivation In Vivo. Clin Infect Dis, 61 (1), pp. 120-128. | Show Abstract | Read more

BACKGROUND: Emerging data relating to human immunodeficiency virus type 1 (HIV-1) cure suggest that vaccination to stimulate the host immune response, particularly cytotoxic cells, may be critical to clearing of reactivated HIV-1-infected cells. However, evidence for this approach in humans is lacking, and parameters required for a vaccine are unknown because opportunities to study HIV-1 reactivation are rare. METHODS: We present observations from a HIV-1 elite controller, not treated with combination antiretroviral therapy, who experienced viral reactivation following treatment for myeloma with melphalan and autologous stem cell transplantation. Mathematical modeling was performed using a standard viral dynamic model. Enzyme-linked immunospot, intracellular cytokine staining, and tetramer staining were performed on peripheral blood mononuclear cells; in vitro CD8 T-cell-mediated control of virion production by autologous CD4 T cells was quantified; and neutralizing antibody titers were measured. RESULTS: Viral rebound was measured at 28,000 copies/mL on day 13 post-transplant before rapid decay to <50 copies/mL in 2 distinct phases with t1/2 of 0.71 days and 4.1 days. These kinetics were consistent with an expansion of cytotoxic effector cells and killing of productively infected CD4 T cells. Following transplantation, innate immune cells, including natural killer cells, recovered with virus rebound. However, most striking was the expansion of highly functional HIV-1-specific cytotoxic CD8 T cells, at numbers consistent with those applied in modeling, as virus control was regained. CONCLUSIONS: These observations provide evidence that the human immune response is capable of controlling coordinated global HIV-1 reactivation, remarkably with potency equivalent to combination antiretroviral therapy. These data will inform design of vaccines for use in HIV-1 curative interventions.

Zhang Y, Makvandi-Nejad S, Qin L, Zhao Y, Zhang T, Wang L, Repapi E, Taylor S et al. 2015. Interferon-induced transmembrane protein-3 rs12252-C is associated with rapid progression of acute HIV-1 infection in Chinese MSM cohort. AIDS, 29 (8), pp. 889-894. | Show Abstract | Read more

BACKGROUND: The interferon-inducible transmembrane protein-3 (IFITM3) is a protein that restricts multiple pathogenic viruses such as influenza virus. The single-nucleotide polymorphism rs12252-C, which is rare in Caucasian populations, but much more common in the Han Chinese population, has been found in much higher homozygous frequency in patients with severe acute influenza. Until now, there has been no study on the effect of this genetic variant on the clinical control of other viral infections. OBJECTIVES: To investigate the impact of IFITM3-rs12252 genotypes on primary HIV-1 infection progression in an acute HIV-1-infected cohort in Beijing (PRIMO), China. DESIGN AND METHODS: We identified IFITM3-rs12252 genotypes of 178 acute HIV-1-infected patients and 196 HIV-negative candidates from the PRIMO cohort. HIV-1 viral load and CD4(+) T-cell counts were monitored at multiple time points during the first year of infection, and the association between IFITM3-rs12252 genotype and disease progression was evaluated. RESULTS: The current study shows that the IFITM3-rs12252 genetic variant affects the progression of HIV-1 infection, but not the acquisition. A significantly higher frequency of the CC/CT genotypes was found in rapid progressors compared to nonprogressors. Patients with CC/CT genotypes showed an elevated peak viremia level and significantly lower CD4(+) T-cell count at multiple time points during the first year of primary infection, and a significantly higher risk of rapid decline of the CD4(+) T-cell count to below 350  cells/μl. CONCLUSION: A novel association between IFITM3 gene polymorphism and rapid disease progression is reported in an acute HIV-1-infected MSM cohort in China.

Hancock G, Yang H, Yorke E, Wainwright E, Bourne V, Frisbee A, Payne TL, Berrong M et al. 2015. Identification of effective subdominant anti-HIV-1 CD8+ T cells within entire post-infection and post-vaccination immune responses. PLoS Pathog, 11 (2), pp. e1004658. | Show Abstract | Read more

Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called "beneficial" regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control.

Peng Y, Wang B, Talaat K, Karron R, Powell TJ, Zeng H, Dong D, Luke CJ, McMichael A, Subbarao K, Dong T. 2015. Boosted Influenza-Specific T Cell Responses after H5N1 Pandemic Live Attenuated Influenza Virus Vaccination. Front Immunol, 6 (JUN), pp. 287. | Show Abstract | Read more

BACKGROUND: In a phase I clinical trial, a H5N1 pandemic live attenuated influenza virus (pLAIV) VN2004 vaccine bearing avian influenza H5N1 hemagglutinin (HA) and NA genes on the A/Ann Arbor cold-adapted vaccine backbone displayed very restricted replication. We evaluated T cell responses to H5N1 pLAIV vaccination and assessed pre-existing T cell responses to determine whether they were associated with restricted replication of the H5N1 pLAIV. METHOD: ELISPOT assays were performed using pools of overlapping peptides spanning the entire H5N1 proteome and the HA proteins of relevant seasonal H1N1 and H3N2 viruses. We tested stored peripheral blood mononuclear cells (PBMCs) from 21 study subjects who received two doses of the H5N1 pLAIV. The PBMCs were collected 1 day before and 7 days after the first and second pLAIV vaccine doses, respectively. RESULT: T cell responses to conserved internal proteins M and NP were significantly boosted by vaccination (p = 0.036). In addition, H5N1 pLAIV appeared to preferentially stimulate and boost pre-existing seasonal influenza virus HA-specific T cell responses that showed low cross-reactivity with the H5 HA. We confirmed this observation by T cell cloning and identified a novel HA-specific epitope. However, we did not find any evidence that pre-existing T cells prevented pLAIV replication and take. CONCLUSION: We found that cross-reactive T cell responses could be boosted by pLAIV regardless of the induction of antibody. The impact of the "original antigenic sin" phenomenon in a subset of volunteers, with preferential expansion of seasonal influenza-specific but not H5N1-specific T cell responses merits further investigation.

Bowles EJ, Schiffner T, Rosario M, Needham GA, Ramaswamy M, McGouran J, Kessler B, LaBranche C et al. 2014. Comparison of neutralizing antibody responses elicited from highly diverse polyvalent heterotrimeric HIV-1 gp140 cocktail immunogens versus a monovalent counterpart in rhesus macaques. PLoS One, 9 (12), pp. e114709. | Show Abstract | Read more

Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a major goal of HIV-1 vaccine design. The challenge is that envelopes (Envs) of circulating viruses are almost certainly different from any Env used in a vaccine. A novel immunogen composed of a highly diverse set of gp140 Envs including subtypes A, B, C, D and F was developed to stimulate a more cross-neutralizing antibody response. Env heterotrimers composed of up to 54 different gp140s were produced with the aim of focusing the response to the conserved regions of Env while reducing the dominance of any individual hypervariable region. Heterotrimeric gp140 Envs of inter- and intra-subtype combinations were shown to bind CD4 and a panel of neutralizing monoclonal antibodies with similar affinity to monovalent UG37 gp140. Macaques immunized with six groups of heterotrimer mixtures showed slightly more potent neutralizing antibody responses in TZM-BL tier 1 and A3R5 tier 2 pseudovirus assays than macaques immunized with monovalent Env gp140, and exhibited a marginally greater focus on the CD4-binding site. Carbopol enhanced neutralization when used as an adjuvant instead of RIBI in combination with UG37 gp140. These data indicate that cross-subtype heterotrimeric gp140 Envs may elicit some improvement of the neutralizing antibody response in macaques compared to monovalent gp140 Env.

Liu D, Zuo T, Hora B, Song H, Kong W, Yu X, Goonetilleke N, Bhattacharya T et al. 2014. Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation Retrovirology, 11 (1), | Show Abstract | Read more

© 2014 Liu et al.; licensee BioMed Central Ltd.Background: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. Results: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. Conclusions: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Liu D, Zuo T, Hora B, Song H, Kong W, Yu X, Goonetilleke N, Bhattacharya T et al. 2014. Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation. Retrovirology, 11 (1), pp. 101. | Show Abstract | Read more

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Ritchie AJ, Cai F, Smith NM, Chen S, Song H, Brackenridge S, Abdool Karim SS, Korber BT, McMichael AJ, Gao F, Goonetilleke N. 2014. Recombination-mediated escape from primary CD8+ T cells in acute HIV-1 infection. Retrovirology, 11 (1), pp. 69. | Show Abstract | Read more

BACKGROUND: A major immune evasion mechanism of HIV-1 is the accumulation of non-synonymous mutations in and around T cell epitopes, resulting in loss of T cell recognition and virus escape. RESULTS: Here we analyze primary CD8+ T cell responses and virus escape in a HLA B*81 expressing subject who was infected with two T/F viruses from a single donor. In addition to classic escape through non-synonymous mutation/s, we also observed rapid selection of multiple recombinant viruses that conferred escape from T cells specific for two epitopes in Nef. CONCLUSIONS: Our study shows that recombination between multiple T/F viruses provide greater options for acute escape from CD8+ T cell responses than seen in cases of single T/F virus infection. This process may contribute to the rapid disease progression in patients infected by multiple T/F viruses.

Campion SL, Brodie TM, Fischer W, Korber BT, Rossetti A, Goonetilleke N, McMichael AJ, Sallusto F. 2014. Proteome-wide analysis of HIV-specific naive and memory CD4(+) T cells in unexposed blood donors. J Exp Med, 211 (7), pp. 1273-1280. | Show Abstract | Read more

The preexisting HIV-1-specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4(+) T cell repertoire in healthy HIV-1-negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4(+) T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1-unexposed, seronegative donors. HIV-1-specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4(+) T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8-12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1-specific helper cell responses by vaccination.

Crawshaw A, Kendrick YR, McMichael AJ, Ho LP. 2014. Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL-10 and suppress T-cell proliferation in sarcoidosis. Eur J Immunol, 44 (7), pp. 2165-2174. | Show Abstract | Read more

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.

McMichael AJ, Koff WC. 2014. Vaccines that stimulate T cell immunity to HIV-1: the next step. Nat Immunol, 15 (4), pp. 319-322. | Show Abstract | Read more

The search for a vaccine against human immunodeficiency virus type 1 (HIV-1) has many hurdles to overcome. Ideally, the stimulation of both broadly neutralizing antibodies and cell-mediated immune responses remains the best option, but no candidate in clinical trials at present has elicited such antibodies, and efficacy trials have not demonstrated any benefit for vaccines designed to stimulate immune responses of CD8(+) T cells. Findings obtained with the simian immunodeficiency virus (SIV) monkey model have provided new evidence that stimulating effective CD8(+) T cell immunity could provide protection, and in this Perspective we explore the path forward for optimizing such responses in humans.

Cole SL, Benam KH, McMichael AJ, Ho LP. 2014. Involvement of the 4-1BB/4-1BBL pathway in control of monocyte numbers by invariant NKT cells. J Immunol, 192 (8), pp. 3898-3907. | Show Abstract | Read more

4-1BB is expressed on invariant (i)NKT cells, but its role is unclear. We showed previously that iNKT cells are involved in control of monocyte numbers during influenza A virus (IAV) infection and now question the role of the 4-1BB costimulatory pathway in the cross-talk between these cells. We found that iNKT cells and monocytes interact to promote expression of 4-1BB and 4-1BBL, respectively. Blockade of 4-1BB/L pathway under resting coculture conditions increased apoptosis of iNKT cells and monocytes. However, activation of iNKT cells overrides this survival signal, causing marked apoptosis of monocytes independent of 4-1BB/L. Blocking 4-1BBL in alpha-galactosylceramide-activated iNKT-monocyte cocultures reduced iNKT proliferation and abrogated monocytic IL-12 production. In vivo, expression of 4-1BB and 4-1BBL is increased on iNKT cells and Ly6C(hi) monocytes, respectively, during IAV infection, and there were lower frequencies of apoptosing Ly6C(hi) monocytes in the blood of iNKT knockout mice and higher numbers of monocytes in lungs compared with infected wild-type mice. Adoptive transfer of iNKT cells into the lungs of these mice reduced lung Ly6C(hi) monocytes levels, even when iNKT cells were preincubated with 4-1BB blocking Abs. These findings suggest that under resting conditions, 4-1BB/L engagement during iNKT-monocyte interaction promotes survival of these cells. When iNKT cells are activated, whether by alpha-galactosylceramide or during IAV infection, iNKT cells induced apoptosis of monocytes via a 4-1BB/L-independent mechanism, reducing monocyte numbers. 4-1BB/L costimulation amplified monocyte-mediated proliferation of iNKT cells, indirectly providing a method for monocytes to control their own numbers during infection.

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Borthwick N, Ahmed T, Ondondo B, Hayes P, Rose A, Ebrahimsa U, Hayton EJ, Black A et al. 2014. Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1 Molecular Therapy, 22 (2), pp. 464-475. | Show Abstract | Read more

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4 + cells and inhibited HIV-1 replication by up to 5.79 log 10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8 + T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro. © The American Society of Gene & Cell Therapy.

Unanue ER, McMichael AJ. 2014. Ita Askonas and her influence in the field of antigen presentation. Curr Opin Immunol, 26 (1), pp. 111-114. | Show Abstract | Read more

This volume on antigen presentation is dedicated to Brigitte Askonas. We summarize here her many contributions to immunology and the impact that her career had on many of us. Critical experimental work on antigen presentation was done in her laboratory under her direction, first examining responses to protein antigens, later examining viruses as she turned her attention to the immunology of infections.

Huang KY, Li CK, Clutterbuck E, Chui C, Wilkinson T, Gilbert A, Oxford J, Lambkin-Williams R, Lin TY, McMichael AJ, Xu XN. 2014. Virus-specific antibody secreting cell, memory B-cell, and sero-antibody responses in the human influenza challenge model. J Infect Dis, 209 (9), pp. 1354-1361. | Show Abstract | Read more

BACKGROUND:  Antibodies play a major role in the protection against influenza virus in human. However, the antibody level is usually short-lived and the cellular mechanisms underlying influenza virus-specific antibody response to acute infection remain unclear. METHODS:  We studied the kinetics and magnitude of influenza virus-specific B-cell and serum antibody responses in relation to virus replication during the course of influenza infection in healthy adult volunteers who were previously seronegative and experimentally infected with seasonal influenza H1N1 A/Brisbane/59/07 virus. RESULTS:  Our data demonstrated a robust expansion of the virus-specific antibody-secreting cells (ASCs) and memory B cells in the peripheral blood, which correlated with both the throat viral load and the duration of viral shedding. The ASC response was obviously detected on day 7 post-infection when the virus was completely cleared from nasal samples, and serum hemagglutination-inhibition antibodies were still undetectable. On day 28 postinfection, influenza virus-specific B cells were further identified from the circulating compartment of isotype-switched B cells. CONCLUSIONS: Virus-specific ASCs could be the earliest marker of B-cell response to a new flu virus infection, such as H7N9 in humans.

Song H, Hora B, Bhattacharya T, Goonetilleke N, Liu MK, Wiehe K, Li H, Iyer SS, McMichael AJ, Perelson AS, Gao F. 2014. Reversion and T cell escape mutations compensate the fitness loss of a CD8+ T cell escape mutant in their cognate transmitted/founder virus. PLoS One, 9 (7), pp. e102734. | Show Abstract | Read more

Immune escape mutations that revert back to the consensus sequence frequently occur in newly HIV-1-infected individuals and have been thought to render the viruses more fit. However, their impact on viral fitness and their interaction with other immune escape mutations have not been evaluated in the background of their cognate transmitted/founder (T/F) viral genomes. To precisely determine the role of reversion mutations, we introduced reversion mutations alone or together with CD8+ T cell escape mutations in their unmodified cognate T/F viral genome and determined their impact on viral fitness in primary CD4+ T cells. Two reversion mutations, V247I and I64T, were identified in Gag and Tat, respectively, but neither had measurable effect on the fitness of their cognate T/F virus. The V247I and G248A mutations that were detected before and concurrently with the potent T cell escape mutation T242N, respectively, were selected by early T cell responses. The V247I or the G248A mutation alone partially restored the fitness loss caused by the T242N mutation. Together they could fully restore the fitness of the T242N mutant to the T/F level. These results demonstrate that the fitness loss caused by a T cell escape mutation could be compensated by preexisting or concurrent reversion and other T cell escape mutations. Our findings indicate that the overall viral fitness is modulated by the complex interplay among T cell escape, compensatory and reversion mutations to maintain the balance between immune escape and viral replication capacity.

McMichael AJ, Haynes BF. 2013. Influenza vaccines: mTOR inhibition surprisingly leads to protection. Nat Immunol, 14 (12), pp. 1205-1207. | Show Abstract | Read more

Mice immunized with influenza virus in the presence of rapamycin, which blocks the formation of germinal centers, make mostly IgM antibodies that protect against infection with multiple subtypes of influenza virus, including avian viruses. © 2013 Nature America, Inc.

Borthwick N, Ahmed T, Ondondo B, Hayes P, Rose A, Ebrahimsa U, Hayton EJ, Black A et al. 2014. Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1. Mol Ther, 22 (2), pp. 464-475. | Show Abstract | Read more

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4(+) cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8(+) T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro.

Rai MA, Zhang Y, Yindom LM, Holmes J, Yu LM, Hao C, Rostron T, Yan H et al. 2013. HLA correlates in a cohort of slow progressors from China: effects on HIV-1 disease progression. AIDS, 27 (17), pp. 2822-2824. | Show Abstract | Read more

We looked at our HIV + slow progressors cohort to determine if there were any human leukocyte antigen (HLA) correlates for protection. No statistically significant allelic differences were found between the HIV + and control cohorts using regression analysis, though trends were noted. Data for Elite Controllers showed an increased frequency of B*57. Likewise, no correlation was inferred with the clinical data of the HIV + cohort. We hypothesize that the protective effect of HLA alleles may have been lost over time.

Goonetilleke N, McMichael AJ. 2013. Immunology. Antigen processing takes a new direction. Science, 340 (6135), pp. 937-938. | Show Abstract | Read more

A vaccine that protects against SIV in monkeys elicits a new kind of T cell response.

Goonetilleke N, McMichael AJ. 2013. HIV-1 vaccines: let's get physical. Immunity, 38 (3), pp. 410-413. | Show Abstract | Read more

Ferguson et al. (2013) use applied physics to quantitate the fitness of HIV-1 Gag based on sequence variability across the protein. This enables a new approach to vaccine design that focuses CD8+ T cell responses on fitness-constrained parts of Gag.

McMichael AJ. 2013. In pursuit of an HIV vaccine: an interview with Andrew McMichael. BMC Biol, 11 pp. 60. | Read more

Powell TJ, Peng Y, Berthoud TK, Blais ME, Lillie PJ, Hill AV, Rowland-Jones SL, McMichael AJ, Gilbert SC, Dong T. 2013. Examination of influenza specific T cell responses after influenza virus challenge in individuals vaccinated with MVA-NP+M1 vaccine. PLoS One, 8 (5), pp. e62778. | Show Abstract | Read more

Current influenza vaccines stimulate neutralising antibody to the haemagglutinin antigen but as there is antigenic drift in HA it is difficult to prepare a vaccine in advance against an emergent strain. A potential strategy is to induce CD8(+) and CD4(+) T cells that recognize epitopes within internal proteins that are less subject to antigenic drift. Augmenting humoral responses to HA with T cell responses to more conserved antigens may result in a more broadly protective vaccine. In this study, we evaluate the quality of influenza specific T cell responses in a clinical trial using MVA-NP+M1 vaccination followed by influenza virus challenge. In vaccinated volunteers, the expression of Granzyme A, Perforin and CD57 on influenza HLA A*02 M158-66 antigen specific cells was higher than non-vaccinated volunteers before and after challenge despite a similar frequency of antigen specific cells. BCL2 expression was lower in vaccinated volunteers. These data indicate that antigen specific T cells are a useful additional measure for use in human vaccination or immunization studies.

Lane J, McLaren PJ, Dorrell L, Shianna KV, Stemke A, Pelak K, Moore S, Oldenburg J et al. 2013. A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A. Hum Mol Genet, 22 (9), pp. 1903-1910. | Show Abstract | Read more

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

Zhao Y, Zhang YH, Denney L, Young D, Powell TJ, Peng YC, Li N, Yan HP et al. 2012. High levels of virus-specific CD4+ T cells predict severe pandemic influenza A virus infection. Am J Respir Crit Care Med, 186 (12), pp. 1292-1297. | Show Abstract | Read more

RATIONALE: T-cell responses have been implicated in control and exacerbation of lung injury during influenza A virus (IAV) infection. OBJECTIVES: To examine the breadth and magnitude of influenza-specific CD4(+) and CD8(+) T-cell responses during acute phase of infection. METHODS: Influenza-specific T-cell response to the entire pandemic H1N1/09 IAV proteome and T cell-related cytokine levels were measured in blood from previously healthy individuals with mild (n = 32) and severe (n = 16) IAV infection during the 2009 influenza pandemic. Virus-specific T-cell response in lung and blood was also performed in two acutely infected, severely ill patients using fluorescent-conjugated pdmH1N1/09 Matrix-MHC-I tetrameric complexes. MEASUREMENTS AND MAIN RESULTS: Strong and broad CD4(+) but not CD8(+) T-cell responses were observed in the blood, and were higher in those with severe disease. Antigen-specific CD8(+) T cells in the lungs were on average 45-fold higher compared with blood in severely ill patients. Paradoxically, in patients with severe disease, IL-17, IL-2, IL-4, and IFN-γ levels were significantly decreased. CONCLUSIONS: High levels of circulating virus-specific CD4(+) T cells to two viral internal proteins (nucleoprotein and matrix) in the first phase of infection are associated with subsequent development of severe IAV infection. This finding could be an early and specific marker for ensuing clinical deterioration. Contrasting levels of antigen-specific CD8(+) T cells in lungs and blood have implications on design and analysis of clinical trials for T-cell vaccines because measurements of T cells in the periphery may not reflect events in the lungs.

Stewart-Jones GB, Simpson P, van der Merwe PA, Easterbrook P, McMichael AJ, Rowland-Jones SL, Jones EY, Gillespie GM. 2012. Structural features underlying T-cell receptor sensitivity to concealed MHC class I micropolymorphisms. Proc Natl Acad Sci U S A, 109 (50), pp. E3483-E3492. | Show Abstract | Read more

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.

Liu MK, Hawkins N, Ritchie AJ, Ganusov VV, Whale V, Brackenridge S, Li H, Pavlicek JW et al. 2013. Vertical T cell immunodominance and epitope entropy determine HIV-1 escape. J Clin Invest, 123 (1), pp. 380-393. | Show Abstract | Read more

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell-mediated in vivo control of HIV-1. Primary HIV-1-specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or "vertical" immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.

McMichael AJ. 2012. Norman Letvin 1949-2012 OBITUARY NATURE IMMUNOLOGY, 13 (9), pp. 801-801.

McMichael AJ. 2012. Norman letvin 1949-2012. Nat Immunol, 13 (9), pp. 801. | Read more

Rajapaksa US, Li D, Peng YC, McMichael AJ, Dong T, Xu XN. 2012. HLA-B may be more protective against HIV-1 than HLA-A because it resists negative regulatory factor (Nef) mediated down-regulation. Proc Natl Acad Sci U S A, 109 (33), pp. 13353-13358. | Show Abstract | Read more

Human leukocyte antigen HLA-B alleles have better protective activity against HIV-1 than HLA-A alleles, possibly due to differences in HLA-restricted HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) function, but the mechanism is unknown. HIV-1 negative regulatory factor (Nef) mediates down-regulation of surface expression of class I HLA (HLA-I) and may therefore impair immune recognition by CTL. Because of sequence differences in the cytoplasmic domains, HLA-A and -B are down-regulated by Nef but HLA-C and -E are not affected. However, the latter are expressed at low levels and are not of major importance in the CTL responses to HIV-1. Here, we compared the role of the cytoplasmic domains of HLA-A and -B in Nef-mediated escape from CTL. We found HLA-B cytoplasmic domains were more resistant to Nef-mediated down-regulation than HLA-A cytoplasmic domains and demonstrated that these differences affect CTL recognition of virus-infected cells in vitro. We propose that the relative resistance to Nef-mediated down-regulation by the cytoplasmic domains of HLA-B compared with HLA-A contributes to the better control of HIV-1 infection associated with HLA-B-restricted CTLs.

Denney L, Kok WL, Cole SL, Sanderson S, McMichael AJ, Ho LP. 2012. Activation of invariant NKT cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of Ly6Chi inflammatory monocyte to M2 macrophages and improved outcome. J Immunol, 189 (2), pp. 551-557. | Show Abstract | Read more

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.

Freel SA, Picking RA, Ferrari G, Ding H, Ochsenbauer C, Kappes JC, Kirchherr JL, Soderberg KA et al. 2012. Initial HIV-1 antigen-specific CD8+ T cells in acute HIV-1 infection inhibit transmitted/founder virus replication. J Virol, 86 (12), pp. 6835-6846. | Show Abstract | Read more

CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8(+) T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8(+) responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8(+) T cells during AHI. Autologous and heterologous CD8(+) T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8(+) T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8(+) antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8(+)-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8(+) T cell-mediated inhibition of virus replication. CD8(+) T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.

Riou C, Ganusov VV, Campion S, Mlotshwa M, Liu MK, Whale VE, Goonetilleke N, Borrow P et al. 2012. Distinct kinetics of Gag-specific CD4+ and CD8+ T cell responses during acute HIV-1 infection. J Immunol, 188 (5), pp. 2198-2206. | Show Abstract | Read more

HIV infection is characterized by a gradual deterioration of immune function, mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4(+) and CD8(+) T cell responses in 12 subtype C-infected individuals with different disease-progression profiles, ranging from acute to chronic HIV infection. The frequencies of Gag-responsive CD4(+) and CD8(+) T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFN-γ(+)CD4(+) T cells was observed at a median of 28 d (interquartile range: 21-81 d) post-Fiebig I/II staging, whereas Gag-specific IFN-γ(+)CD8(+) T cell responses peaked at a median of 253 d (interquartile range: 136-401 d) and showed a significant biphasic expansion. The proportion of TNF-α-expressing cells within the IFN-γ(+)CD4(+) T cell population increased (p = 0.001) over time, whereas TNF-α-expressing cells within IFN-γ(+)CD8(+) T cells declined (p = 0.005). Both Gag-responsive CD4(+) and CD8(+) T cells showed decreased Ki67 expression within the first 120 d post-Fiebig I/II staging. Prior to the disappearance of Gag-responsive Ki67(+)CD4(+) T cells, these cells positively correlated (p = 0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8(+) T cell compartment. Overall, these observations indicated that circulating Gag-responsive CD4(+) and CD8(+) T cell frequencies and functions are not synchronous, and properties change rapidly at different tempos during early HIV infection.

Simpson PD, Moysi E, Wicks K, Sudan K, Rowland-Jones SL, McMichael AJ, Knight J, Gillespie GM. 2012. Functional differences exist between TNFα promoters encoding the common -237G SNP and the rarer HLA-B*5701-linked A variant. PLoS One, 7 (7), pp. e40100. | Show Abstract | Read more

A large body of functional and epidemiological evidence have previously illustrated the impact of specific MHC class I subtypes on clinical outcome during HIV-1 infection, and these observations have recently been re-iterated in genome wide association studies (GWAS). Yet because of the complexities surrounding GWAS-based approaches and the lack of knowledge relating to the identity of rarer single nucleotide polymorphism (SNP) variants, it has proved difficult to discover independent causal variants associated with favourable immune control. This is especially true of the candidate variants within the HLA region where many of the recently proposed disease influencing SNPs appear to reflect linkage with 'protective' MHC class I alleles. Yet causal MHC-linked SNPs may exist but remain overlooked owing to the complexities associated with their identification. Here we focus on the ancestral TNFα promoter -237A variant (rs361525), shown historically to be in complete linkage disequilibrium with the 'protective' HLA-B*5701 allele. Many of the ancestral SNPs within the extended TNFα promoter have been associated with both autoimmune conditions and disease outcomes, however, the direct role of these variants on TNFα expression remains controversial. Yet, because of the important role played by TNFα in HIV-1 infection, and given the proximity of the -237 SNP to the core promoter, its location within a putative repressor region previously characterized in mice, and its disruption of a methylation-susceptible CpG dinucleotide motif, we chose to carefully evaluate its impact on TNFα production. Using a variety of approaches we now demonstrate that carriage of the A SNP is associated with lower TNFα production, via a mechanism not readily explained by promoter methylation nor the binding of transcription factors or repressors. We propose that the -237A variant could represent a minor causal SNP that additionally contributes to the HLA-B*5701-mediated 'protective' effect during HIV-1 infection.

Cited:

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Wilkinson TM, Li CKF, Chui CSC, Huang AKY, Perkins M, Liebner JC, Lambkin-Williams R, Gilbert A et al. 2012. Preexisting influenza-specific CD4 + T cells correlate with disease protection against influenza challenge in humans Nature Medicine, 18 (2), pp. 274-280. | Show Abstract | Read more

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4 +, but not CD8 +, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4 + cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains. © 2012 Nature America, Inc. All rights reserved. © 2012 Nature America, Inc. All rights reserved.

Ritchie AJ, Kuldanek K, Moodie Z, Wang ZM, Fox J, Nsubuga RN, Legg K, Birabwa EF et al. 2012. Comparison of sexual behavior and HIV risk between two HIV-1 serodiscordant couple cohorts: the CHAVI 002 study. PLoS One, 7 (5), pp. e37727. | Show Abstract | Read more

BACKGROUND: The CHAVI002 study was designed to characterize immune responses, particularly HIV-specific T-cell responses, amongst 2 cohorts of HIV-exposed seronegative (HESN) individuals. The absence of a clear definition of HESNs has impaired comparison of research within and between such cohorts. This report describes two distinct HESN cohorts and attempts to quantify HIV exposure using a 'HIV risk index' (RI) model. METHODS: HIV serodiscordant couples (UK; 24, Uganda; 72) and HIV unexposed seronegative (HUSN) controls (UK; 14, Uganda; 26 couples, 3 individuals) completed sexual behavior questionnaires every 3 months over a 9 month period. The two cohorts were heterogeneous, with most HESNs in the UK men who have sex with men (MSM), while all HESNs in Uganda were in heterosexual relationships. Concordance of responses between partners was determined. Each participant's sexual behavior score (SBS) was estimated based on the number and type of unprotected sex acts carried out in defined time periods. Independent HIV acquisition risk factors (partner plasma viral load, STIs, male circumcision, pregnancy) were integrated with the SBS, generating a RI for each HESN. RESULTS: 96 HIV serodiscordant couples completed 929 SBQs. SBSs remained relatively stable amongst the UK cohort, whilst decreasing from Visit 1 to 2 in the Ugandan cohort. Compared to the Ugandan cohort, SBSs and RIs in the UK cohort were lower at visit 1, and generally higher at later visits. Differences between the cohorts, with lower rates of ART use in Uganda and higher risk per-act sex in the UK, had major impacts on the SBSs and RIs of each cohort. There was one HIV transmission event in the UK cohort. CONCLUSIONS: Employment of a risk quantification model facilitated quantification and comparison of HIV acquisition risk across two disparate HIV serodiscordant couple cohorts.

McMichael AJ, Haynes BF. 2012. Lessons learned from HIV-1 vaccine trials: new priorities and directions. Nat Immunol, 13 (5), pp. 423-427. | Show Abstract | Read more

A vaccine against human immunodeficiency virus (HIV) seems to be on the horizon. Correlates of risk of infection for [corrected] the RV144 vaccine trial have been found. There is understanding of what makes HIV envelope-specific antibodies broadly neutralizing and new T cell vaccine approaches can overcome virus variability.

Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM et al. 2012. IFITM3 restricts the morbidity and mortality associated with influenza Nature,

Wilkinson TM, Li CK, Chui CS, Huang AK, Perkins M, Liebner JC, Lambkin-Williams R, Gilbert A et al. 2012. Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans. Nat Med, 18 (2), pp. 274-280. | Show Abstract | Read more

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.

Cited:

26

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Rosario M, Borthwick N, Stewart-Jones GB, Mbewe-Mvula A, Bridgeman A, Colloca S, Montefiori D, McMichael AJ et al. 2012. Prime-boost regimens with adjuvanted synthetic long peptides elicit T cells and antibodies to conserved regions of HIV-1 in macaques AIDS, 26 (3), pp. 275-284. | Show Abstract | Read more

Objectives: Administration of synthetic long peptides (SLPs) derived from human papillomavirus to cervical cancer patients resulted in clinical benefit correlated with expansions of tumour-specific T cells. Because vaginal mucosa is an important port of entry for HIV-1, we have explored SLP for HIV-1 vaccination. Using immunogen HIVconsv derived from the conserved regions of HIV-1, we previously showed in rhesus macaques that SLP.HIVconsv delivered as a boost increased the breath of T-cell specificities elicited by single-gene vaccines. Here, we compared and characterized the use of electroporated pSG2.HIVconsv DNA (D) and imiquimod/montanide-adjuvanted SLP.HIVconsv (S) as priming vaccines for boosting with attenuated chimpanzee adenovirus ChAdV63.HIVconsv (C) and modified vaccinia virus Ankara MVA.HIVconsv (M). Design: Prime-boost regimens of DDDCMS, DSSCMS and SSSCMS in rhesus macaques. Methods: Animals' blood was analysed regularly throughout the vaccination for HIV-1-specific T-cell and antibody responses. Results: We found that electroporation spares DNA dose, both SLP.HIVconsv and pSG2.HIVconsv DNA primed weakly HIVconsv-specific T cells, regimen DDDCM induced the highest frequencies of oligofunctional, proliferating CD4 and CD8 T cells, and a subsequent SLP.HIVconsv boost expanded primarily CD4 cells. DSS was the most efficient regimen inducing antibodies binding to regions of trimeric HIV-1 Env, which are highly conserved among the four major global clades, although no unequivocal neutralizing activity was detected. Conclusion: The present results encourage evaluation of the SLP.HIVconsv vaccine modality in human volunteers along the currently trialled pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines. These results are discussed in the context of the RV144 trial outcome. © 2012 Wolters Kluwer Health Lippincott Williams & Wilkins.

Hanke T, McMichael AJ. 2011. HIV-1: from escapism to conservatism. Eur J Immunol, 41 (12), pp. 3390-3393. | Read more

Rosario M, Borthwick N, Stewart-Jones GB, Mbewe-Mvula A, Bridgeman A, Colloca S, Montefiori D, McMichael AJ et al. 2012. Prime-boost regimens with adjuvanted synthetic long peptides elicit T cells and antibodies to conserved regions of HIV-1 in macaques. AIDS, 26 (3), pp. 275-284. | Show Abstract | Read more

OBJECTIVES: Administration of synthetic long peptides (SLPs) derived from human papillomavirus to cervical cancer patients resulted in clinical benefit correlated with expansions of tumour-specific T cells. Because vaginal mucosa is an important port of entry for HIV-1, we have explored SLP for HIV-1 vaccination. Using immunogen HIVconsv derived from the conserved regions of HIV-1, we previously showed in rhesus macaques that SLP.HIVconsv delivered as a boost increased the breath of T-cell specificities elicited by single-gene vaccines. Here, we compared and characterized the use of electroporated pSG2.HIVconsv DNA (D) and imiquimod/montanide-adjuvanted SLP.HIVconsv (S) as priming vaccines for boosting with attenuated chimpanzee adenovirus ChAdV63.HIVconsv (C) and modified vaccinia virus Ankara MVA.HIVconsv (M). DESIGN: Prime-boost regimens of DDDCMS, DSSCMS and SSSCMS in rhesus macaques. METHODS: Animals' blood was analysed regularly throughout the vaccination for HIV-1-specific T-cell and antibody responses. RESULTS: We found that electroporation spares DNA dose, both SLP.HIVconsv and pSG2.HIVconsv DNA primed weakly HIVconsv-specific T cells, regimen DDDCM induced the highest frequencies of oligofunctional, proliferating CD4(+) and CD8(+) T cells, and a subsequent SLP.HIVconsv boost expanded primarily CD4(+) cells. DSS was the most efficient regimen inducing antibodies binding to regions of trimeric HIV-1 Env, which are highly conserved among the four major global clades, although no unequivocal neutralizing activity was detected. CONCLUSION: The present results encourage evaluation of the SLP.HIVconsv vaccine modality in human volunteers along the currently trialled pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines. These results are discussed in the context of the RV144 trial outcome.

Powell TJ, Fox A, Peng Y, Quynh Mai LET, Lien VT, Hang NL, Wang L, Lee LY et al. 2012. Identification of H5N1-specific T-cell responses in a high-risk cohort in vietnam indicates the existence of potential asymptomatic infections. J Infect Dis, 205 (1), pp. 20-27. | Show Abstract | Read more

BACKGROUND: Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam. METHODS: Peripheral blood mononuclear cells were tested using interferon-γ enzyme-linked immunospot T assays measuring the response to peptides of influenza H5, H3, and H1 hemagglutinin (HA), N1 and N2 neuraminidase, and the internal proteins of H3N2. Horse erythrocyte hemagglutination inhibition assay was performed to detect antibodies against H5N1. RESULTS: Twenty-four of 747 individuals demonstrated H5-specific T-cell responses but little or no cross-reactivity with H3 or H1 HA peptides. H5N1 peptide-specific T-cell lines that did not cross-react with H1 or H3 influenza virus HA peptides were generated. Four individuals also had antibodies against H5N1. CONCLUSIONS: This is the first report of ex vivo H5 HA-specific T-cell responses in a healthy but H5N1-exposed population. Our results indicate that the presence of H5N1-specific T cells could be an additional diagnostic tool for asymptomatic H5N1 infection.

Pelak K, Need AC, Fellay J, Shianna KV, Feng S, Urban TJ, Ge D, De Luca A et al. 2011. Copy number variation of KIR genes influences HIV-1 control. PLoS Biol, 9 (11), pp. e1001208. | Show Abstract | Read more

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

Kok WL, Denney L, Benam K, Cole S, Clelland C, McMichael AJ, Ho LP. 2012. Pivotal Advance: Invariant NKT cells reduce accumulation of inflammatory monocytes in the lungs and decrease immune-pathology during severe influenza A virus infection. J Leukoc Biol, 91 (3), pp. 357-368. | Show Abstract | Read more

Little is known of how a strong immune response in the lungs is regulated to minimize tissue injury during severe influenza A virus (IAV) infection. Here, using a model of lethal, high-pathogenicity IAV infection, we first show that Ly6C(hi)Ly6G(-) inflammatory monocytes, and not neutrophils, are the main infiltrate in lungs of WT mice. Mice devoid of iNKT cells (Jα18(-/-) mice) have increased levels of inflammatory monocytes, which correlated with increased lung injury and mortality (but not viral load). Activation of iNKT cells correlated with reduction of MCP-1 levels and improved outcome. iNKT cells were able to selectively lyse infected, MCP-1-producing monocytes in vitro, in a CD1d-dependent process. Our study provides a detailed profile and kinetics of innate immune cells in the lungs during severe IAV infection, highlighting inflammatory monocytes as the major infiltrate and identifying a role for iNKT cells in control of these cells and lung immune-pathology.

Ganusov VV, Goonetilleke N, Liu MK, Ferrari G, Shaw GM, McMichael AJ, Borrow P, Korber BT, Perelson AS. 2011. Fitness costs and diversity of the cytotoxic T lymphocyte (CTL) response determine the rate of CTL escape during acute and chronic phases of HIV infection. J Virol, 85 (20), pp. 10518-10528. | Show Abstract | Read more

HIV-1 often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single-genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes, we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, an increase in the number of epitope-specific CTL responses can reduce the rate of viral escape from a given epitope-specific CTL response, particularly if CD8+ T cells compete for killing of infected cells or control virus replication nonlytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL responses that need to be induced by vaccination to reduce (or even prevent) viral escape following HIV infection.

Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM. 2011. Phenotypic Analysis of Antigen-Specific T Lymphocytes (Reprinted from Science, vol 274, pg 94-96, 1996) JOURNAL OF IMMUNOLOGY, 187 (1),

Cohen MS, Shaw GM, McMichael AJ, Haynes BF. 2011. Acute HIV-1 Infection. N Engl J Med, 364 (20), pp. 1943-1954. | Read more

Ranasinghe SRF, Kramer HB, Wright C, Kessler BM, di Gleria K, Zhang Y, Gillespie GM, Blais M-E et al. 2011. The Antiviral Efficacy of HIV-Specific CD8(+) T-Cells to a Conserved Epitope Is Heavily Dependent on the Infecting HIV-1 Isolate PLOS PATHOGENS, 7 (5), | Show Abstract | Read more

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef90-97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A-H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses. © 2011 Ranasinghe et al.

Im EJ, Hong JP, Roshorm Y, Bridgeman A, Létourneau S, Liljeström P, Potash MJ, Volsky DJ, McMichael AJ, Hanke T. 2011. Protective efficacy of serially up-ranked subdominant CD8+ T cell epitopes against virus challenges. PLoS Pathog, 7 (5), pp. e1002041. | Show Abstract | Read more

Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans.

Brackenridge S, Evans EJ, Toebes M, Goonetilleke N, Liu MK, di Gleria K, Schumacher TN, Davis SJ, McMichael AJ, Gillespie GM. 2011. An early HIV mutation within an HLA-B*57-restricted T cell epitope abrogates binding to the killer inhibitory receptor 3DL1. J Virol, 85 (11), pp. 5415-5422. | Show Abstract | Read more

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

Ritchie AJ, Campion SL, Kopycinski J, Moodie Z, Wang ZM, Pandya K, Moore S, Liu MK et al. 2011. Differences in HIV-specific T cell responses between HIV-exposed and -unexposed HIV-seronegative individuals. J Virol, 85 (7), pp. 3507-3516. | Show Abstract | Read more

HIV-1-specific T lymphocyte responses in individuals exposed to HIV-1 but who remain persistently seronegative (HESNs) have been reported in some but not all previous studies. This study was designed to resolve unequivocally the question of whether HESNs make HIV-1-specific T cell responses. We performed a blind investigation to measure HIV-1-specific T cell responses in both HIV-1-serodiscordant couples and HIV-1-unexposed seronegative controls (HUSNs). We found low-frequency HIV-1-specific T cells in both HESNs and HUSNs but show that the response rates were higher over time in the former (P = 0.01). Furthermore, the magnitudes of the HIV-1-specific T cell responses were significantly higher among responding HESNs than among HUSNs over time (P = 0.002). In both groups, responses were mediated by CD4 T cells. The responses were mapped to single peptides, which often corresponded to epitopes restricted by multiple HLA-DR types that have previously been detected in HIV-1-infected patients. HIV-1-specific T cell responses in HUSNs and some HESNs likely represent cross-reactivity to self or foreign non-HIV-1 antigens. The significantly greater T cell responses in HESNs, including in two who were homozygous for CCR5Δ32, demonstrates that HIV-1-specific T cell responses can be induced or augmented by exposure to HIV-1 without infection.

Benam KH, Kok WL, McMichael AJ, Ho LP. 2011. Alternative spliced CD1d transcripts in human bronchial epithelial cells. PLoS One, 6 (8), pp. e22726. | Show Abstract | Read more

CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity.

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Cohen MS, Shaw GM, McMichael AJ, Haynes BF. 2011. Acute HIV-1 Infection New England Journal of Medicine, 364 (20), pp. 1943-1954. | Read more

Ferrari G, Korber B, Goonetilleke N, Liu MK, Turnbull EL, Salazar-Gonzalez JF, Hawkins N, Self S et al. 2011. Relationship between functional profile of HIV-1 specific CD8 T cells and epitope variability with the selection of escape mutants in acute HIV-1 infection. PLoS Pathog, 7 (2), pp. e1001273. | Show Abstract | Read more

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants.

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22

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Wu J, Zhong X, Li CKF, Zhou JF, Lu M, Huang KY, Dong M, Liu Y et al. 2011. Optimal vaccination strategies for 2009 pandemic H1N1 and seasonal influenza vaccines in humans Vaccine, 29 (5), pp. 1009-1016. | Show Abstract | Read more

A randomized clinical trial was conducted to assess whether the immunogenicity of seasonal and pandemic (H1N1/09) influenza vaccines is affected by the order of vaccine administration. 151 healthy adult volunteers were randomized into three groups. All groups received one dose (15 μg haemagglutinin) each of a pandemic H1N1 vaccine and a seasonal trivalent vaccine. Group 1 received the pandemic H1N1 vaccine first, followed by the seasonal vaccine 21 days later. Group 2 received vaccinations in vice versa and Group 3 received both vaccines simultaneously. Post-vaccination blood samples were collected to determine the immunogenicity by hemagglutination-inhibition (HI), microneutralization (MN), and B cell ELISPOT assays. All three vaccination strategies were well-tolerated and generated specific immune responses. However, we found a significant difference in magnitude of antibody responses to pandemic H1N1 between the three groups. Pre- or co-vaccination with the seasonal flu vaccine led to a significant reduction by 50% in HI titre to pandemic H1N1 virus after pandemic vaccination. Pre- or co-vaccination of pandemic H1N1 vaccine had no effect on seasonal flu vaccination. MN and ELISPOT assays showed a similar effect. Vaccination with pandemic H1N1 vaccine first is recommended to avoid an associated inhibitory effect by the seasonal trivalent flu vaccine. Clinical_Trials identifier: NCT01008137. © 2010.

Campion S, Cohen MS, McMichael AJ, Galvin S, Goonetilleke N. 2011. Improved detection of latent Mycobacterium tuberculosis infection in HIV-1 seropositive individuals using cultured cellular assays. Eur J Immunol, 41 (1), pp. 255-257. | Read more

Petrovski S, Fellay J, Shianna KV, Carpenetti N, Kumwenda J, Kamanga G, Kamwendo DD, Letvin NL et al. 2011. Common human genetic variants and HIV-1 susceptibility: a genome-wide survey in a homogeneous African population. AIDS, 25 (4), pp. 513-518. | Show Abstract | Read more

OBJECTIVE: To date, CCR5 variants remain the only human genetic factors to be confirmed to impact HIV-1 acquisition. However, protective CCR5 variants are largely absent in African populations, in which sporadic resistance to HIV-1 infection is still unexplained. We investigated whether common genetic variants associate with HIV-1 susceptibility in Africans. METHODS: We performed a genome-wide association study (GWAS) in a population of 1532 individuals from Malawi, a country with high prevalence of HIV-1 infection. Using single-nucleotide polymorphisms (SNPs) present on the genome-wide chip, we also investigated previously reported associations with HIV-1 susceptibility or acquisition. Recruitment was coordinated by the Center for HIV/AIDS Vaccine Immunology at two sexually transmitted infection clinics. HIV status was determined by HIV rapid tests and nucleic acid testing. RESULTS: After quality control, the population consisted of 848 high-risk seronegative and 531 HIV-1 seropositive individuals. Logistic regression testing in an additive genetic model was performed for SNPs that passed quality control. No single SNP yielded a significant P value after correction for multiple testing. The study was sufficiently powered to detect markers with genotype relative risk 2.0 or more and minor allele frequencies 12% or more. CONCLUSION: This is the first GWAS of host determinants of HIV-1 susceptibility, performed in an African population. The absence of any significant association can have many possible explanations: rarer genetic variants or common variants with weaker effect could be responsible for the resistance phenotype; alternatively, resistance to HIV-1 infection might be due to nongenetic parameters or to complex interactions between genes, immunity and environment.

McMichael AJ, Jones EY. 2010. Genetics. First-class control of HIV-1. Science, 330 (6010), pp. 1488-1490. | Show Abstract | Read more

Genome-wide association studies reveal amino acids of the major histocompatibility complex that associate with the rate of progression to AIDS.

Campion S, Cohen MS, McMichael AJ, Galvin S, Goonetilleke N. 2011. Improved detection of latent Mycobacterium tuberculosis infection in HIV-1 seropositive individuals using cultured cellular assays. Eur J Immunol, 41 (1), pp. 255-257. | Read more

Sattentau QJ, McMichael AJ. 2010. The Literature SCIENTIST, 24 (10), pp. 65-66.

Rosario M, Bridgeman A, Quakkelaar ED, Quigley MF, Hill BJ, Knudsen ML, Ammendola V, Ljungberg K et al. 2010. Long peptides induce polyfunctional T cells against conserved regions of HIV-1 with superior breadth to single-gene vaccines in macaques. Eur J Immunol, 40 (7), pp. 1973-1984. | Show Abstract | Read more

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.

Shu Y, Li CK, Li Z, Gao R, Liang Q, Zhang Y, Dong L, Zhou J et al. 2010. Avian influenza A(H5N1) viruses can directly infect and replicate in human gut tissues. J Infect Dis, 201 (8), pp. 1173-1177. | Show Abstract | Read more

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.

Lockstone HE, Sanderson S, Kulakova N, Baban D, Leonard A, Kok WL, McGowan S, McMichael AJ, Ho LP. 2010. Gene set analysis of lung samples provides insight into pathogenesis of progressive, fibrotic pulmonary sarcoidosis. Am J Respir Crit Care Med, 181 (12), pp. 1367-1375. | Show Abstract | Read more

RATIONALE: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome. OBJECTIVES: To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes. METHODS: We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7). MEASUREMENTS AND MAIN RESULTS: Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF). CONCLUSIONS: The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis.

Cella M, Presti R, Vermi W, Lavender K, Turnbull E, Ochsenbauer-Jambor C, Kappes JC, Ferrari G et al. 2010. Loss of DNAM-1 contributes to CD8+ T-cell exhaustion in chronic HIV-1 infection. Eur J Immunol, 40 (4), pp. 949-954. | Show Abstract | Read more

The hallmark of chronic viral infections is a progressive exhaustion of antigen-specific CD8(+) T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8(+) T cells that makes them dysfunctional. Here, we show that during human chronic HIV-1 infection, a CD8(+) T-cell positive costimulatory pathway mediated by DNAX-activating molecule-1 is also disrupted. Thus, DNAX-activating molecule-1 downregulation on CD8(+) T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection.

Goonetilleke N, Liu MK, Ganusov V, Giorgi E, Salazar J, Li H, Kirchner J, Turnbull E et al. 2010. The Role of T Cell Immunity in the Control of HIV Infection INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 14 pp. E19-E20. | Read more

Scherer EM, Leaman DP, Zwick MB, McMichael AJ, Burton DR. 2010. Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions. Proc Natl Acad Sci U S A, 107 (4), pp. 1529-1534. | Show Abstract | Read more

The broadly neutralizing anti-HIV antibody 4E10 recognizes an epitope very close to the virus membrane on the glycoprotein gp41. It was previously shown that epitope recognition improves in a membrane context and that 4E10 binds directly, albeit weakly, to lipids. Furthermore, a crystal structure of Fab 4E10 complexed to an epitope peptide revealed that the centrally placed, protruding H3 loop of the antibody heavy chain does not form peptide contacts. To investigate the hypothesis that the H3 loop apex might interact with the viral membrane, two Trp residues in this region were substituted separately or in combination with either Ala or Asp by site-directed mutagenesis. The resultant IgG variants exhibited similar affinities for an epitope peptide as WT 4E10 but lower apparent affinities for both viral membrane mimetic liposomes and Env(-) virus. Variants also exhibited lower apparent affinities for Env(+) virions and failed to significantly neutralize a number of 4E10-sensitive viruses. For the extremely sensitive HXB2 virus, variants did neutralize, but at 37- to >250-fold lower titers than WT 4E10, with Asp substitutions exerting a greater effect on neutralization potency than Ala substitutions. Because reductions in lipid binding reflect trends in neutralization potency, we conclude that Trp residues in the antibody H3 loop enable membrane proximal epitope recognition through favorable lipid interactions. The requirement for lipophilic residues such as Trp adjacent to the antigen binding site may explain difficulties in eliciting 4E10-like neutralizing antibody responses by immunization and helps define a unique motif for antibody recognition of membrane proximal antigens.

Wu J, Zhong X, Li CK, Zhou JF, Lu M, Huang KY, Dong M, Liu Y et al. 2011. Optimal vaccination strategies for 2009 pandemic H1N1 and seasonal influenza vaccines in humans. Vaccine, 29 (5), pp. 1009-1016. | Show Abstract | Read more

A randomized clinical trial was conducted to assess whether the immunogenicity of seasonal and pandemic (H1N1/09) influenza vaccines is affected by the order of vaccine administration. 151 healthy adult volunteers were randomized into three groups. All groups received one dose (15 μg haemagglutinin) each of a pandemic H1N1 vaccine and a seasonal trivalent vaccine. Group 1 received the pandemic H1N1 vaccine first, followed by the seasonal vaccine 21 days later. Group 2 received vaccinations in vice versa and Group 3 received both vaccines simultaneously. Post-vaccination blood samples were collected to determine the immunogenicity by hemagglutination-inhibition (HI), microneutralization (MN), and B cell ELISPOT assays. All three vaccination strategies were well-tolerated and generated specific immune responses. However, we found a significant difference in magnitude of antibody responses to pandemic H1N1 between the three groups. Pre- or co-vaccination with the seasonal flu vaccine led to a significant reduction by 50% in HI titre to pandemic H1N1 virus after pandemic vaccination. Pre- or co-vaccination of pandemic H1N1 vaccine had no effect on seasonal flu vaccination. MN and ELISPOT assays showed a similar effect. Vaccination with pandemic H1N1 vaccine first is recommended to avoid an associated inhibitory effect by the seasonal trivalent flu vaccine.

Sattentau QJ, McMichael AJ. 2010. New templates for HIV-1 antibody-based vaccine design F1000 Biology Reports, 2 (1), | Show Abstract | Read more

A current strategy for the design of neutralizing antibody-based vaccines to prevent HIV-1 transmission is that of reverse engineering, starting from a neutralizing antibody and working back to reconstruct its epitope by structure-based design technology. However, the field has been impeded by a lack of appropriate antibodies for use as templates. Recently, new antibodies have been described that may fulfil this role, invigorating the field. © 2010 Faculty of 1000 Ltd.

Kramer HB, Lavender KJ, Qin L, Stacey AR, Liu MK, di Gleria K, Simmons A, Gasper-Smith N et al. 2010. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection. PLoS Pathog, 6 (5), pp. e1000893. | Show Abstract | Read more

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

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Kramer HB, Lavender KJ, Qin L, Stacey AR, Liu MKP, di Gleria K, Simmons A, Gasper-Smith N et al. 2010. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection PLoS Pathogens, 6 (5), pp. 1-12. | Show Abstract | Read more

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, b-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies. © 2010 Kramer et al.

Denney L, Aitken C, Li CK, Wilson-Davies E, Kok WL, Clelland C, Rooney K, Young D et al. 2010. Reduction of natural killer but not effector CD8 T lymphocytes in three consecutive cases of severe/lethal H1N1/09 influenza A virus infection. PLoS One, 5 (5), pp. e10675. | Show Abstract | Read more

BACKGROUND: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We present the cellular immunology profile in the blood, and detailed clinical (and post-mortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. CONCLUSION/SIGNIFICANCE: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype.

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Denney L, Aitken C, Li CKF, Wilson-Davies E, Kok WL, Clelland C, Rooney K, Young D et al. 2010. Reduction of natural killer but not effector CD8 t lymphoyctes in three consecutive cases of severe/lethal H1N1/09 influenza a virus infection PLoS ONE, 5 (5), | Show Abstract | Read more

Background: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. Methodology/Principal Findings: We present the cellular immunology profile in the blood, and detailed clinical (and postmortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. Conclusion/Significance: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype. © 2010 Denney et al.

McMichael AJ, Borrow P, Tomaras GD, Goonetilleke N, Haynes BF. 2010. The immune response during acute HIV-1 infection: clues for vaccine development. Nat Rev Immunol, 10 (1), pp. 11-23. | Show Abstract | Read more

The early immune response to HIV-1 infection is likely to be an important factor in determining the clinical course of disease. Recent data indicate that the HIV-1 quasispecies that arise following a mucosal infection are usually derived from a single transmitted virus. Moreover, the finding that the first effective immune responses drive the selection of virus escape mutations provides insight into the earliest immune responses against the transmitted virus and their contributions to the control of acute viraemia. Strong innate and adaptive immune responses occur subsequently but they are too late to eliminate the infection. In this Review, we discuss recent studies on the kinetics and quality of early immune responses to HIV-1 and their implications for developing a successful preventive HIV-1 vaccine.

Fischer W, Ganusov VV, Giorgi EE, Hraber PT, Keele BF, Leitner T, Han CS, Gleasner CD et al. 2010. Transmission of single HIV-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing. PLoS One, 5 (8), pp. e12303. | Show Abstract | Read more

We used ultra-deep sequencing to obtain tens of thousands of HIV-1 sequences from regions targeted by CD8+ T lymphocytes from longitudinal samples from three acutely infected subjects, and modeled viral evolution during the critical first weeks of infection. Previous studies suggested that a single virus established productive infection, but these conclusions were tempered because of limited sampling; now, we have greatly increased our confidence in this observation through modeling the observed earliest sample diversity based on vastly more extensive sampling. Conventional sequencing of HIV-1 from acute/early infection has shown different patterns of escape at different epitopes; we investigated the earliest escapes in exquisite detail. Over 3-6 weeks, ultradeep sequencing revealed that the virus explored an extraordinary array of potential escape routes in the process of evading the earliest CD8 T-lymphocyte responses--using 454 sequencing, we identified over 50 variant forms of each targeted epitope during early immune escape, while only 2-7 variants were detected in the same samples via conventional sequencing. In contrast to the diversity seen within epitopes, non-epitope regions, including the Envelope V3 region, which was sequenced as a control in each subject, displayed very low levels of variation. In early infection, in the regions sequenced, the consensus forms did not have a fitness advantage large enough to trigger reversion to consensus amino acids in the absence of immune pressure. In one subject, a genetic bottleneck was observed, with extensive diversity at the second time point narrowing to two dominant escape forms by the third time point, all within two months of infection. Traces of immune escape were observed in the earliest samples, suggesting that immune pressure is present and effective earlier than previously reported; quantifying the loss rate of the founder virus suggests a direct role for CD8 T-lymphocyte responses in viral containment after peak viremia. Dramatic shifts in the frequencies of epitope variants during the first weeks of infection revealed a complex interplay between viral fitness and immune escape.

Fellay J, Ge D, Shianna KV, Colombo S, Ledergerber B, Cirulli ET, Urban TJ, Zhang K et al. 2009. Common genetic variation and the control of HIV-1 in humans. PLoS Genet, 5 (12), pp. e1000791. | Show Abstract | Read more

To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians.

Kulakova N, Urban B, McMichael AJ, Ho LP. 2010. Functional analysis of dendritic cell-T cell interaction in sarcoidosis. Clin Exp Immunol, 159 (1), pp. 82-86. | Show Abstract | Read more

The primary cause of the intense immune response in sarcoidosis is unclear. Potentially, a functional abnormality in dendritic cells (DCs) could cause a reduction in clearance of antigen and downstream persistence in immune activity. In this study, we investigate the interaction between monocyte-derived dendritic cells and T cells in patients with sarcoidosis compared to normal controls (n = 8 each) by examining the kinetics of autologous and allogeneic mixed leucocyte reactions over 9-10 days. We found markedly depressed proliferation kinetics in autologous DC-peripheral blood mononuclear cell (PBMC) co-cultures from sarcoid patients compared to normal subjects. In allogeneic experiments PBMCs from patients showed a reduced response to allogeneic DCs from a single donor, but no difference was observed in the ability of patients and control DCs to stimulate proliferation of allogeneic PBMC from a single donor. We conclude that there is a markedly impaired autologous mixed leucocyte reaction (MLR) in sarcoidosis patients. In allogeneic MLR, monocyte-derived DCs in sarcoidosis were able to stimulate T cells normally, but PBMCs responses were reduced. This contradicts recent published studies on ex vivo isolated myeloid DCs from sarcoidosis patients although, potentially, an in vivo conditioning factor, which reduces DC function in sarcoidosis, could be a unifying explanation for the contrasting findings.

Urban TJ, Weintrob AC, Fellay J, Colombo S, Shianna KV, Gumbs C, Rotger M, Pelak K et al. 2009. CCL3L1 and HIV/AIDS susceptibility. Nat Med, 15 (10), pp. 1110-1112. | Read more

Roshorm Y, Hong JP, Kobayashi N, McMichael AJ, Volsky DJ, Potash MJ, Takiguchi M, Hanke T. 2009. Novel HIV-1 clade B candidate vaccines designed for HLA-B*5101(+) patients protected mice against chimaeric ecotropic HIV-1 challenge. Eur J Immunol, 39 (7), pp. 1831-1840. | Show Abstract | Read more

Novel candidate HIV-1 vaccines have been constructed, which are tailor-designed for HLA-B*5101(+) patients infected with HIV-1 clade B. These vaccines employ novel immunogen HIVB-B*5101 derived from consensus HIV-1 clade B Gag p17 and p24 regions coupled to two Pol-derived B*5101-restricted epitopes, which are together with a third B*5101 epitope in Gag dominant in HIV-1-infected long-term non-progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB-B*5101 immunogen in cultured cells. Heterologous DNA prime-recombinant MVA boost regimen induced efficiently HIV-1-specific CD8(+) T-cell responses in BALB/c mice. These vaccine-elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV-1/NL4-3 and ecotropic HIV-1/NDK chimaeric viruses with HIV-1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti-HIV-1 gp120 antibodies. These results support further development of HIVB-B*5101 vaccines in combined heterologous-modality regimens. The use of allele-specific vaccines in humans is discussed in the context of other developments in the HIV-1 field.

Goonetilleke N, Liu MK, Salazar-Gonzalez JF, Ferrari G, Giorgi E, Ganusov VV, Keele BF, Learn GH et al. 2009. The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection. J Exp Med, 206 (6), pp. 1253-1272. | Show Abstract | Read more

Identification of the transmitted/founder virus makes possible, for the first time, a genome-wide analysis of host immune responses against the infecting HIV-1 proteome. A complete dissection was made of the primary HIV-1-specific T cell response induced in three acutely infected patients. Cellular assays, together with new algorithms which identify sites of positive selection in the virus genome, showed that primary HIV-1-specific T cells rapidly select escape mutations concurrent with falling virus load in acute infection. Kinetic analysis and mathematical modeling of virus immune escape showed that the contribution of CD8 T cell-mediated killing of productively infected cells was earlier and much greater than previously recognized and that it contributed to the initial decline of plasma virus in acute infection. After virus escape, these first T cell responses often rapidly waned, leaving or being succeeded by T cell responses to epitopes which escaped more slowly or were invariant. These latter responses are likely to be important in maintaining the already established virus set point. In addition to mutations selected by T cells, there were other selected regions that accrued mutations more gradually but were not associated with a T cell response. These included clusters of mutations in envelope that were targeted by NAbs, a few isolated sites that reverted to the consensus sequence, and bystander mutations in linkage with T cell-driven escape.

Murphy RL, Autran B, Katlama C, Brucker G, Debre P, Calvez V, Clotet B, Clumeck N et al. 2009. A step ahead on the HIV collaboratory. Science, 324 (5932), pp. 1264-1265. | 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.

Walter BL, Armitage AE, Graham SC, de Oliveira T, Skinhøj P, Jones EY, Stuart DI, McMichael AJ, Chesebro B, Iversen AK. 2009. Functional characteristics of HIV-1 subtype C compatible with increased heterosexual transmissibility. AIDS, 23 (9), pp. 1047-1057. | Show Abstract | Read more

BACKGROUND: Despite the existence of over 50 subtypes and circulating recombinant forms of HIV-1, subtype C dominates the heterosexual pandemic causing approximately 56% of all infections. OBJECTIVE: To evaluate whether viral genetic factors may contribute to the observed subtype-C predominance. METHODS: Chimeric viruses were generated using V1-V3 envelope fragments from a subtype-A/C dually infected woman with preferential genital replication of subtype C. Viral adaptation, spread and cell fusion ability were evaluated in vitro using peripheral blood mononuclear cells and HeLa-CD4-CCR5 cell lines, sequencing and cloning. Structural modeling was performed using a crystal structure of gp120-CD4-X5. Phylogenetic analysis was done using subtype-A, subtype-B and subtype-C sequences from blood and cervix of 37 infected women and database sequences. RESULTS: We identified two envelope motifs, compact V1-V2 loops and V3-316T, which are found at high frequency throughout subtype-C evolution and affect gp120 interactions with CD4 and CCR5, respectively. When a V1-Delta5 deletion or V3-A316T was incorporated into subtype A, each increased viral fusion and spread several fold in peripheral blood mononuclear cell and cell lines with low CCR5 expression. Structural modeling suggested the formation of an additional hydrogen bond between V3 and CCR5. Moreover, we found preferential selection of HIV with 316T and/or extremely short V1-V2 loops in cervices of three women infected with subtypes A/C, B or C. CONCLUSION: As CD4-CCR5-T cells are key targets for genital HIV infection and cervical selection can favor compact V1-V2 loops and 316T, which increase viral infectivity, we propose that these conserved subtype-C motifs may contribute to transmission and spread of this subtype.

Walley NM, Julg B, Dickson SP, Fellay J, Ge D, Walker BD, Carrington M, Cohen MS et al. 2009. The Duffy antigen receptor for chemokines null promoter variant does not influence HIV-1 acquisition or disease progression. Cell Host Microbe, 5 (5), pp. 408-410. | Read more

Winstone N, Guimarães-Walker A, Roberts J, Brown D, Loach V, Goonetilleke N, Hanke T, McMichael AJ. 2009. Increased detection of proliferating, polyfunctional, HIV-1-specific T cells in DNA-modified vaccinia virus Ankara-vaccinated human volunteers by cultured IFN-gamma ELISPOT assay. Eur J Immunol, 39 (4), pp. 975-985. | Show Abstract | Read more

Induction of a long-term immunological memory, which can expand and defend the host upon pathogen encounter, is the "holy grail" of vaccinology. Here, using a sensitive cultured IFN-gamma ELISPOT assay, we show that 50% (15 out of 30) of healthy, HIV-1/2-uninfected volunteers who received pTHr.HIVA DNA prime-modified vaccinia virus Ankara. HIVA boost vaccine regimen 1 to 3 1/2 years ago had detectable HIV-1-specific T-cell responses. These T cells, predominantly of the CD4(+) subtype, could proliferate and produce multiple cytokines in response to in vitro peptide stimulation. Peptide mapping studies showed that the vaccine-induced CD4(+) T cells were mostly directed toward epitopes targeted in HIV-1-infected individuals. In addition, we used the same assay to re-evaluate 51 volunteers from past vaccine trial IAVI-006 and corrected the previously reported 10% of vaccine responders to 50%. Thus, we confirmed that cultured assays are a valuable tool for studying T-cell memory. These results are discussed in the context of the current state-of-affairs of the HIV-1 vaccine field.

Ranasinghe SR, Kramer HB, Wright C, Kessler BM, di Gleria K, Zhang Y, Gillespie GM, Blais ME et al. 2011. The antiviral efficacy of HIV-specific CD8⁺ T-cells to a conserved epitope is heavily dependent on the infecting HIV-1 isolate. PLoS Pathog, 7 (5), pp. e1001341. | Show Abstract | Read more

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef₉₀₋₉₇ epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A-H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.

Yang H, Yan H, Li W, Zhang X, Fischer W, Zhang H, Wu H, Korber BT, McMichael AJ, Xu X, Goonetilleke N. 2009. A greater breadth of HIV-1-specific T cell responses detected using mosaic peptides compare to consensus peptides RETROVIROLOGY, 6

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

Liu MK, Ferrari G, Salazar J, Keele B, Tanner RL, Hraber P, Giorgi E, Ganusov VV et al. 2009. The role of early T-cell responses in subjects with acute HIV-1 infection RETROVIROLOGY, 6 (SUPPL. 3),

Teoh D, Johnson LA, Hanke T, McMichael AJ, Jackson DG. 2009. Blocking development of a CD8+ T cell response by targeting lymphatic recruitment of APC. J Immunol, 182 (4), pp. 2425-2431. | Show Abstract | Read more

Generating a protective immune response to viral infection is known to depend upon the priming and clonal expansion of virus-specific CD8(+) T cells by Ag-loaded dendritic cells (DC) within secondary lymphoid tissue. However, the actual initiation of the response involves critical upstream events that control the recruitment of mature Ag-charged DC from the periphery via afferent lymphatics, events that are still only partly understood. Recent evidence has revealed that transmigration of lymphatic endothelium by DC is regulated by the adhesion molecules ICAM-1 and VCAM-1 both in vitro and in vivo. These findings imply that lymphatic entry may be an important rate-limiting step in primary immunity and a possible target for immune intervention. In this study, we have explored such possibilities using an F(5) TCR-transgenic mouse model to assess the contribution of lymphatic cell adhesion molecules in the CD8(+) T cell response to influenza virus nucleoprotein (NP). We show for the first time that immunization with ICAM-1- and VCAM-1-blocking mAbs can impair the T cell response in lymph node-draining sites of dermally administered nucleoprotein vaccine (MVA.HIVA.NP) by targeting lymphatic uptake of Ag-loaded DC ahead of other cell adhesion molecule-dependent events. These results reveal lymphatic entry as an important step that may be rate limiting in the development of immunity and reconfirm its potential as a target for localized immunotherapy in inflammation and tissue rejection.

Bangs SC, Baban D, Cattan HJ, Li CK, McMichael AJ, Xu XN. 2009. Human CD4+ memory T cells are preferential targets for bystander activation and apoptosis. J Immunol, 182 (4), pp. 1962-1971. | Show Abstract | Read more

There is much evidence that T cells may be activated via mechanisms that act independently of direct TCR ligation. Despite this, the question of whether such forms of bystander T cell activation occur during immune responses is hotly debated. To address some outstanding questions, we set up an in vitro system within which to analyze bystander T cell activation in human T cells, in the absence of the possibility for TCR cross-reactivity. In addition, we have investigated the genetic, phenotypic, and functional characteristics of bystander-activated T cells. In this study, we show that bystander T cell activation is, indeed, observed during a specific immune response, and that it occurs preferentially among CD4(+) memory T cells. Furthermore, bystander-activated T cells display a distinct gene expression profile. The mechanism for bystander T cell activation involves soluble factors, and the outcome is an elevated level of apoptosis. This may provide an explanation for the attrition of T cell memory pools of heterologous specificity during immune responses to pathogens such as viruses.

Li CK, Wu H, Yan H, Ma S, Wang L, Zhang M, Tang X, Temperton NJ et al. 2008. T cell responses to whole SARS coronavirus in humans. J Immunol, 181 (8), pp. 5490-5500. | Show Abstract

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-gamma ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8(+) T cell responses were more frequent and of a greater magnitude than CD4(+) T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27(+)/CD45RO(+)) with a significantly higher frequency of polyfunctional CD4(+) T cells producing IFN-gamma, TNF-alpha, and IL-2, and CD8(+) T cells producing IFN-gamma, TNF-alpha, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.

Lee LY, Ha DOLA, Simmons C, de Jong MD, Chau NV, Schumacher R, Peng YC, McMichael AJ et al. 2008. Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest, 118 (10), pp. 3478-3490. | Show Abstract | Read more

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Armitage AE, Katzourakis A, de Oliveira T, Welch JJ, Belshaw R, Bishop KN, Kramer B, McMichael AJ, Rambaut A, Iversen AK. 2008. Conserved footprints of APOBEC3G on Hypermutated human immunodeficiency virus type 1 and human endogenous retrovirus HERV-K(HML2) sequences. J Virol, 82 (17), pp. 8743-8761. | Show Abstract | Read more

The human polynucleotide cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are antiviral restriction factors capable of inducing extensive plus-strand guanine-to-adenine (G-to-A) hypermutation in a variety of retroviruses and retroelements, including human immunodeficiency virus type 1 (HIV-1). They differ in target specificity, favoring plus-strand 5'GG and 5'GA dinucleotide motifs, respectively. To characterize their mutational preferences in detail, we analyzed single-copy, near-full-length HIV-1 proviruses which had been hypermutated in vitro by hA3G or hA3F. hA3-induced G-to-A mutation rates were significantly influenced by the wider sequence context of the target G. Moreover, hA3G, and to a lesser extent hA3F, displayed clear tetranucleotide preference hierarchies, irrespective of the genomic region examined and overall hypermutation rate. We similarly analyzed patient-derived hypermutated HIV-1 genomes using a new method for estimating reference sequences. The majority of these, regardless of subtype, carried signatures of hypermutation that strongly correlated with those induced in vitro by hA3G. Analysis of genome-wide hA3-induced mutational profiles confirmed that hypermutation levels were reduced downstream of the polypurine tracts. Additionally, while hA3G mutations were found throughout the genome, hA3F often intensely mutated shorter regions, the locations of which varied between proviruses. We extended our analysis to human endogenous retroviruses (HERVs) from the HERV-K(HML2) family, finding two elements that carried clear footprints of hA3G activity. This constitutes the most direct evidence to date for hA3G activity in the context of natural HERV infections, demonstrating the involvement of this restriction factor in defense against retroviral attacks over millions of years of human evolution.

Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, Chang C, Harndahl M et al. 2008. Antigen processing shapes HIV-specific CTL-response hierarchies WIENER KLINISCHE WOCHENSCHRIFT, 120 pp. 146-146.

Armitage AE, McMichael AJ, Drakesmith H. 2008. Reflecting on a quarter century of HIV research. Nat Immunol, 9 (8), pp. 823-826. | Read more

Ho LP, Denney L, Luhn K, Teoh D, Clelland C, McMichael AJ. 2008. Activation of invariant NKT cells enhances the innate immune response and improves the disease course in influenza A virus infection. Eur J Immunol, 38 (7), pp. 1913-1922. | Show Abstract | Read more

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection.

Simon AK, Newsom-Davis T, Frayne ME, Ch'en PF, McMichael AJ, Screaton GR. 2008. Generation of tumour-rejecting anti-carbohydrate monoclonal antibodies using melanoma modified with Fas ligand. Int Immunol, 20 (4), pp. 525-534. | Show Abstract | Read more

Carbohydrate antigens such as glycolipids and glycoproteins are over-expressed in a variety of cancers and have therefore been identified as ideal candidates for tumour vaccines. Detection of anti-carbohydrate antibodies is associated with a good prognosis in cancer patients. However, generation of an efficient adaptive immune response has been hampered by the low immunogenicity of carbohydrates due to tolerance. Here, we describe a method by which tumour-rejecting antibodies directed against carbohydrates can be elicited in two different melanoma mouse models. Thus, using the murine melanoma B16F10 over-expressing Fas ligand (FasL), we have generated mAbs against cancer carbohydrate antigens expressed by the melanoma. Importantly, passive transfer of mAbs resulted in rejection of melanoma in vivo. Their protective effect in vivo was dependent on FcR and in vitro antibody-dependent cellular phagocytosis. They were also able to delay tumour growth when injected after the tumour was established. FasL-expressing tumours as an adjuvant are a novel way to generate anti-carbohydrate antibodies able to reject tumours in vivo.

Duvall MG, Precopio ML, Ambrozak DA, Jaye A, McMichael AJ, Whittle HC, Roederer M, Rowland-Jones SL, Koup RA. 2008. Polyfunctional T cell responses are a hallmark of HIV-2 infection. Eur J Immunol, 38 (2), pp. 350-363. | Show Abstract | Read more

HIV-2 is distinguished clinically and immunologically from HIV-1 infection by delayed disease progression and maintenance of HIV-specific CD4(+) T cell help in most infected subjects. Thus, HIV-2 provides a unique natural human model in which to investigate correlates of immune protection against HIV disease progression. Here, we report a detailed assessment of the HIV-2-specific CD4(+) and CD8(+) T cell response compared to HIV-1, using polychromatic flow cytometry to assess the quality of the HIV-specific T cell response by measuring IFN-gamma, IL-2, TNF-alpha, MIP-1beta, and CD107a mobilization (degranulation) simultaneously following Gag peptide stimulation. We find that HIV-2-specific CD4(+) and CD8(+) T cells are more polyfunctional that those specific for HIV-1 and that polyfunctional HIV-2-specific T cells produce more IFN-gamma and TNF-alpha on a per-cell basis than monofunctional T cells. Polyfunctional HIV-2-specific CD4(+) T cells were generally more differentiated and expressed CD57, while there was no association between function and phenotype in the CD8(+) T cell fraction. Polyfunctional HIV-specific T cell responses are a hallmark of non-progressive HIV-2 infection and may be related to good clinical outcome in this setting.

Ishizuka J, Stewart-Jones GB, van der Merwe A, Bell JI, McMichael AJ, Jones EY. 2008. The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vbeta domain. Immunity, 28 (2), pp. 171-182. | Show Abstract | Read more

Immunodominant and public T cell receptor (TCR) usage is relatively common in many viral diseases yet surprising in the context of the large naive TCR repertoire. We examined the highly conserved Vbeta17:Valpha10.2 JM22 T cell response to the influenza matrix peptide (58-66)-HLA-A*0201 (HLA-A2-flu) through extensive kinetic, thermodynamic, and structural analyses. We found several conformational adjustments that accompany JM22-HLA-A2-flu binding and identified a binding "hotspot" within the Vbeta domain of the TCR. Within this hotspot, key germline-encoded CDR1 and CDR2 loop residues and a crucial but commonly coded residue in the hypervariable region of CDR3 provide the basis for the substantial bias in the selection of the germline-encoded Vbeta17 domain. The chances of having a substantial number of T cells in the naive repertoire that have HLA-A2-flu-specific Vbeta17 receptors may consequently be relatively high, thus explaining the immunodominant usage of this clonotype.

Li D, Chen N, McMichael AJ, Screaton GR, Xu XN. 2008. Generation and characterisation of CD1d tetramer produced by a lentiviral expression system. J Immunol Methods, 330 (1-2), pp. 57-63. | Show Abstract | Read more

The alpha-galactosylceramide (alphaGalCer)-loaded CD1d tetramer remains the most powerful tool in identifying natural killer T (NKT) cells, a subpopulation of T cells that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. The difficulty of generating large amounts of the alphaGalCer-CD1d tetramer has limited its availability and consequently hampered the study of NKT cells. In this report, we used a lentiviral system to generate stable cell lines producing beta2m-CD1d single chain protein in large quantities and in a relatively short period of time. When the protein was loaded with alphaGalCer and tetramerised with fluorescence-labelled streptavidin, its ability to efficiently bind to NKT cells was confirmed both by phenotype analysis and functional study. The CD1d tetramer generated from these stable cell lines should facilitate a wide range of studies on the biology and clinical applications of CD1d-restricted NKT cells.

Duvall MG, Loré K, Blaak H, Ambrozak DA, Adams WC, Santos K, Geldmacher C, Mascola JR et al. 2007. Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection. J Virol, 81 (24), pp. 13486-13498. | Show Abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4(+) T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4(+) T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4(+) T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4(+) T cells. Despite this, we found that HIV-2-specific CD4(+) T cells contained more viral DNA than memory CD4(+) T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4(+) T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4(+) T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4(+) T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4(+) T-cell help in vivo.

McMichael AJ. 2007. Triple bypass: complicated paths to HIV escape. J Exp Med, 204 (12), pp. 2785-2788. | Show Abstract | Read more

Human immunodeficiency virus (HIV) type 1 is highly efficient at evading immune responses and persisting, ultimately causing fatal immunodeficiency in some patients. Mutation in the epitopes recognized by cytolytic CD8+ T cells (CTLs) is one such escape process. A new study now shows that one HIV-1 escape mutation may also result in impaired dendritic cell (DC) activity, possibly impairing later T cell responses to the same and other epitopes. The new data complete our understanding of the mechanisms by which the CTL response to an immunodominant gag epitope presented by human histocompatibility leukocyte antigen (HLA)-B27 is evaded. The complexity of the full escape helps to explain why patients with this HLA type progress to AIDS more slowly than average.

Schneidewind A, Brockman MA, Yang R, Adam RI, Li B, Le Gall S, Rinaldo CR, Craggs SL et al. 2007. Escape from the dominant HLA-B27-restricted cytotoxic T-lymphocyte response in Gag is associated with a dramatic reduction in human immunodeficiency virus type 1 replication. J Virol, 81 (22), pp. 12382-12393. | Show Abstract | Read more

Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8(+) T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R(264)K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R(264)K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R(264)K mutation was associated in vivo with the development of a rare secondary mutation, S(173)A, which restored viral replication in vitro. Furthermore, infectivity of the R(264)K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R(264)K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R(264)K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.

McMichael AJ. 2007. From influenza to HIV--and back? Nat Immunol, 8 (11), pp. 1149-1151. | Read more

Fellay J, Shianna KV, Ge D, Colombo S, Ledergerber B, Weale M, Zhang K, Gumbs C et al. 2007. A whole-genome association study of major determinants for host control of HIV-1. Science, 317 (5840), pp. 944-947. | Show Abstract | Read more

Understanding why some people establish and maintain effective control of HIV-1 and others do not is a priority in the effort to develop new treatments for HIV/AIDS. Using a whole-genome association strategy, we identified polymorphisms that explain nearly 15% of the variation among individuals in viral load during the asymptomatic set-point period of infection. One of these is found within an endogenous retroviral element and is associated with major histocompatibility allele human leukocyte antigen (HLA)-B*5701, whereas a second is located near the HLA-C gene. An additional analysis of the time to HIV disease progression implicated two genes, one of which encodes an RNA polymerase I subunit. These findings emphasize the importance of studying human genetic variation as a guide to combating infectious agents.

Cox JH, McMichael AJ, Screaton GR, Xu XN. 2007. CTLs target Th cells that acquire bystander MHC class I-peptide complex from APCs. J Immunol, 179 (2), pp. 830-836. | Show Abstract

CTLs can acquire MHC class I-peptide complexes from their target cells, whereas CD4(+) T cells obtain MHC class II-peptide complexes from APCs in a TCR-specific manner. As a consequence, Ag-specific CTL can kill each other (fratricide) or CD4(+) T cells become APCs themselves. The purpose of the acquisition is not fully understood and may be either inhibition or prolongation of an immunological response. In this study, we demonstrate that human CD4(+) Th cells are able to capture membrane fragments from APC during the process of immunological synapse formation. The fragments contain not only MHC class II-peptide complexes but also MHC class I-peptide complexes, rendering these cells susceptible to CTL killing in an Ag-specific manner. The control of CD4(+) Th cells by Ag-specific CTL, therefore, maybe another mechanism to regulate CD4(+) T cell expansion in normal immune responses or cause immunopathology during the course of viral infections such as HIV.

Davenport MP, Price DA, McMichael AJ. 2007. The T cell repertoire in infection and vaccination: implications for control of persistent viruses. Curr Opin Immunol, 19 (3), pp. 294-300. | Show Abstract | Read more

Most currently available vaccines target acute infectious agents such as polio, smallpox and influenza virus. The development of vaccines to persistent infectious agents has proven much more difficult. Although it is possible to generate T cell responses to such viruses, these responses are currently unable to prevent the establishment of infection, and immune control may be lost during the chronic phase. Recent advances have increased our understanding of the interactions between persistent viruses and the available T cell repertoire, and will guide approaches to the generation and maintenance of an 'ideal' T cell response.

Lühn K, Simmons CP, Moran E, Dung NT, Chau TN, Quyen NT, Thao LETT, Van Ngoc T et al. 2007. Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J Exp Med, 204 (5), pp. 979-985. | Show Abstract | Read more

Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease.

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Karlsson AC, Iversen AKN, Chapman JM, de Oliveira T, Spotts G, McMichael AJ, Davenport MP, Hecht FM, Nixon DF. 2007. Sequential Broadening of CTL Responses in Early HIV-1 Infection Is Associated with Viral Escape PLOS ONE, 2 (2), pp. e225-e225. | Show Abstract | Read more

Background. Antigen-specific CTL responses are thought to play a central role in containment of HIV-1 infection, but no consistent correlation has been found between the magnitude and/or breadth of response and viral load changes during disease progression. Methods and Findings. We undertook a detailed investigation of longitudinal CTL responses and HIV-1 evolution beginning with primary infection in 11 untreated HLA-A2 positive individuals. A subset of patients developed broad responses, which selected for consensus B epitope variants in Gag, Pol, and Nef, suggesting CTL-induced adaptation of HIV-1 at the population level. The patients who developed viral escape mutations and broad autologous CTL responses over time had a significantly higher increase in viral load during the first year of infection compared to those who did not develop viral escape mutations. Conclusions. A continuous dynamic development of CTL responses was associated with viral escape from temporarily effective immune responses. Our results suggest that broad CTL responses often represent footprints left by viral CTL escape rather than effective immune control, and help explain earlier findings that fail to show an association between breadth of CTL responses and viral load. Our results also demonstrate that CTL pressures help to maintain certain elements of consensus viral sequence, which likely represent viral escape from common HLA-restricted CTL responses. The ability of HIV to evolve to escape CTL responses restricted by a common HLA type highlights the challenges posed to development of an effective CTL-based vaccine. © 2007 Karlsson et al.

Larke N, Im EJ, Wagner R, Williamson C, Williamson AL, McMichael AJ, Hanke T. 2007. Combined single-clade candidate HIV-1 vaccines induce T cell responses limited by multiple forms of in vivo immune interference. Eur J Immunol, 37 (2), pp. 566-577. | Show Abstract | Read more

We assessed in mice whether broad CD8+ T cell responses capable of efficient recognition of multiple HIV-1 clades could be induced using current single-clade vaccine constructs that were or will be used in clinical trials in Europe and Africa. We found that single-clade A, B and C vaccines applied alone induced only limited cross-clade reactivity and that the epitope hierarchy varied according to the immunizing clade. However, combining single-clade HIV-1 vaccines into multi-clade formulations resulted in multiple forms of in vivo immune interference such as original antigenic sin and antagonism, which dampened or even abrogated induction of responses to epitope variants and reduced the breadth of induced T cell responses. Simultaneous administration of individual clade-specific vaccines into anatomically separated sites on the body alleviated antagonism and increased the number of detectable epitope responses. Although cross-reactivity of murine CD8+ T cells does not directly translate to humans, the molecular interactions involved in triggering T cell responses are the same in mouse and man. Thus, these results have important ramifications for the design of both prophylactic and therapeutic vaccines against HIV-1 and other highly variable pathogens.

Chiu C, Heaps AG, Cerundolo V, McMichael AJ, Bangham CR, Callan MF. 2007. Early acquisition of cytolytic function and transcriptional changes in a primary CD8+ T-cell response in vivo. Blood, 109 (3), pp. 1086-1094. | Show Abstract | Read more

Functional studies show that programming of CD8+ T cells occurs early after initial antigen encounter within as little as 2 hours. To define the molecular basis of these events, we transferred TCR transgenic T cells from F5 Rag-/- mice into naive recipients and stimulated them with recombinant vaccinia expressing the immunodominant influenza epitope NP366-374. Transcription in epitope-specific cytotoxic T lymphocytes (CTLs) was analyzed using Affymetrix 430 2.0 GeneChips and quantitative polymerase chain reaction (PCR). We demonstrated an early transcriptional burst with the greatest number of genes reaching peak expression 12 hours after stimulation. Using in vivo cytotoxicity assays we demonstrated that early up-regulation of cytolytic genes was accompanied by acquisition of killing capacity within 24 hours of stimulation. However, T-cell proliferation was not observed until 48 hours. We therefore conclude that clonal expansion rather than acquisition of effector function is the rate-limiting step in the development of a primary CTL response.

Hanke T, Goonetilleke N, McMichael AJ, Dorrell L. 2007. Clinical experience with plasmid DNA- and modified vaccinia virus Ankara-vectored human immunodeficiency virus type 1 clade A vaccine focusing on T-cell induction. J Gen Virol, 88 (Pt 1), pp. 1-12. | Show Abstract | Read more

Candidate human immunodeficiency virus type 1 (HIV-1) vaccines focusing on T-cell induction, constructed as pTHr.HIVA DNA and modified vaccinia virus Ankara (MVA).HIVA, were delivered in a heterologous prime-boost regimen. The vaccines were tested in several hundred healthy or HIV-1-infected volunteers in Europe and Africa. Whilst larger trials of hundreds of volunteers suggested induction of HIV-1-specific T-cell responses in <15 % of healthy vaccinees, a series of small, rapid trials in 12-24 volunteers at a time with a more in-depth analysis of vaccine-elicited T-cell responses proved to be highly informative and provided more encouraging results. These trials demonstrated that the pTHr.HIVA vaccine alone primed consistently weak and mainly CD4(+), but also CD8(+) T-cell responses, and the MVA.HIVA vaccine delivered a consistent boost to both CD4(+) and CD8(+) T cells, which was particularly strong in HIV-1-infected patients. Thus, whilst the search is on for ways to enhance T-cell priming, MVA is a useful boosting vector for human subunit genetic vaccines.

Létourneau S, Im EJ, Mashishi T, Brereton C, Bridgeman A, Yang H, Dorrell L, Dong T, Korber B, McMichael AJ, Hanke T. 2007. Design and pre-clinical evaluation of a universal HIV-1 vaccine. PLoS One, 2 (10), pp. e984. | Show Abstract | Read more

BACKGROUND: One of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test. METHODOLOGY AND FINDINGS: To address the HIV-1 variation, we designed a novel T cell immunogen, designated HIV(CONSV), by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each segment is a consensus sequence from one of the four major HIV-1 clades A, B, C and D, which alternate to ensure equal clade coverage. The gene coding for the HIV(CONSV) protein was inserted into the three most studied vaccine vectors, plasmid DNA, human adenovirus serotype 5 and modified vaccine virus Ankara (MVA), and induced HIV-1-specific T cell responses in mice. We also demonstrated that these conserved regions prime CD8(+) and CD4(+) T cell to highly conserved epitopes in humans and that these epitopes, although usually subdominant, generate memory T cells in patients during natural HIV-1 infection. SIGNIFICANCE: Therefore, this vaccine approach provides an attractive and testable alternative for overcoming the HIV-1 variability, while focusing T cell responses on regions of the virus that are less likely to mutate and escape. Furthermore, this approach has merit in the simplicity of design and delivery, requiring only a single immunogen to provide extensive coverage of global HIV-1 population diversity.

Bangs SC, McMichael AJ, Xu XN. 2006. Bystander T cell activation--implications for HIV infection and other diseases. Trends Immunol, 27 (11), pp. 518-524. | Show Abstract | Read more

T cells are subject to tight regulatory measures, as uncontrolled responses might be detrimental to the host. Control measures include central or thymic tolerance, and peripheral tolerance mechanisms acting after naive T cells have encountered their cognate antigen, such as anergy induction, the contraction phase (whereby the majority of the expanded effector population undergoes apoptosis), and the action of regulatory T (Treg) cells. However, bystander T-cell activation circumvents the requirement for specific T-cell receptor stimulation, enabling T cells to bypass certain control checkpoints. The physiological relevance of the phenomenon is the subject of much controversy. This article argues that although of little consequence in the healthy individual, bystander activation could have a devastating impact in the context of disease. We focus on HIV and infection-triggered autoimmune disease as examples.

Ondondo BO, Yang H, Dong T, di Gleria K, Suttill A, Conlon C, Brown D, Williams P et al. 2006. Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses. Eur J Immunol, 36 (10), pp. 2585-2594. | Show Abstract | Read more

Virus-specific CD4+ T cells with IL-2-secreting and/or proliferative capacity are detected readily in HIV-1-infected long-term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV-1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV-1 gag p24/p17 (MVA.HIVA) on HIV-1-specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)-treated subjects using CD8-depleted IFN-gamma ELISPOT assays, intracellular cytokine staining assays for IL-2 and IFN-gamma, and a CFSE-based proliferation assay. Gag-specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV-uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL-2 or IFN-gamma, alone or simultaneously, were also augmented. These findings indicate that functional virus-specific T helper cells can be boosted by vaccination in chronic HIV-1 infection. Further evaluation of their role in viraemia control is warranted.

Im EJ, Nkolola JP, di Gleria K, McMichael AJ, Hanke T. 2006. Induction of long-lasting multi-specific CD8+ T cells by a four-component DNA-MVA/HIVA-RENTA candidate HIV-1 vaccine in rhesus macaques. Eur J Immunol, 36 (10), pp. 2574-2584. | Show Abstract | Read more

As a part of a long-term effort to develop vaccine against HIV-1 clade A inducing protective T cell responses in humans, we run mutually complementing studies in humans and non-human primates (NHP) with the aim to maximize vaccine immunogenicity. The candidate vaccine under development has four components, pTHr.HIVA and pTH.RENTA DNA, and modified vaccinia virus Ankara (MVA).HIVA and MVA.RENTA, delivered in a heterologous DNA prime-MVA boost regimen. While the HIVA (Gag/epitopes) components have been tested in NHP and over 300 human subjects, we plan to test in humans the RENTA (reverse transcriptase, gp41, Nef, Tat) vaccines designed to broaden HIVA-induced responses in year 2007. Here, we investigated the four-component vaccine long-term immunogenicity in Mamu-A*01-positive rhesus macaques and demonstrated that the vaccine-induced T cells were multi-specific, multi-functional, readily proliferated to recall peptides and were circulating in the peripheral blood of vaccine recipients over 1 year after vaccine administration. The consensus clade A-elicited T cells recognized 50% of tested epitope variants from other HIV-1 clades. Thus, the DNA-MVA/HIVA-RENTA vaccine induced memory T cells of desirable characteristics and similarities to those induced in humans by HIVA vaccines alone; however, single-clade vaccines may not elicit sufficiently cross-reactive responses.

Gillespie GM, Stewart-Jones G, Rengasamy J, Beattie T, Bwayo JJ, Plummer FA, Kaul R, McMichael AJ et al. 2006. Strong TCR conservation and altered T cell cross-reactivity characterize a B*57-restricted immune response in HIV-1 infection. J Immunol, 177 (6), pp. 3893-3902. | Show Abstract

HLA-B*57 is associated with slower disease progression to AIDS, and CD8+ T cell responses to B*57-restricted epitopes are thought to contribute to this protective effect. In this study, we evaluate the B*57-restricted p24 KAFSPEVIPMF (KF11) immune response which is immunodominant during chronic infection. Previously, we observed that the KF11 clade variants KGFNPEVIPMF [A2G,S4N] and KAFNPEIIMPF [S4N,V7I], sharing a position 4 mutation, are differentially recognized by KF11-specific T cells. By combining structural and cellular studies, we now demonstrate that the KF11 and [A2G,S4N] epitopes induce distinct functional responses in [A2G,S4N] and KF11-specific T cells, respectively, despite minimal structural differences between the individual B*57-peptide complexes. Recently, we also elucidated the highly distinct structure of KF11 in complex with B*5703, and have now characterized the CD8+ T cell repertoire recognizing this epitope. We now report striking features of TCR conservation both in terms of TCR Valpha and Vbeta chain usage, and throughout the hypervariable region. Collectively, our findings highlight unusual features of the B*5701/B*5703-KF11-specific immune responses which could influence disease progression and that might be important to consider when designing future vaccine regimens.

Duvall MG, Jaye A, Dong T, Brenchley JM, Alabi AS, Jeffries DJ, van der Sande M, Togun TO et al. 2006. Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection. J Immunol, 176 (11), pp. 6973-6981. | Show Abstract

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57-), and more frequently produced IFN-gamma or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-gamma+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.

Goonetilleke N, Moore S, Dally L, Winstone N, Cebere I, Mahmoud A, Pinheiro S, Gillespie G et al. 2006. Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes. J Virol, 80 (10), pp. 4717-4728. | Show Abstract | Read more

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.

Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 2006. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides (reprinted from Cell, vol. 44, pg. 959-968, yr. 1986) JOURNAL OF IMMUNOLOGY, 176 (9), pp. 5141-5150.

Schaft N, Lankiewicz B, Drexhage J, Berrevoets C, Moss DJ, Levitsky V, Bonneville M, Lee SP et al. 2006. T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNgamma production. Int Immunol, 18 (4), pp. 591-601. | Show Abstract | Read more

EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.

Iversen AK, Stewart-Jones G, Learn GH, Christie N, Sylvester-Hviid C, Armitage AE, Kaul R, Beattie T et al. 2006. Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope. Nat Immunol, 7 (2), pp. 179-189. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Chen N, McCarthy C, Drakesmith H, Li D, Cerundolo V, McMichael AJ, Screaton GR, Xu XN. 2006. HIV-1 down-regulates the expression of CD1d via Nef. Eur J Immunol, 36 (2), pp. 278-286. | Show Abstract | Read more

HIV-1 has evolved several strategies to subvert host immune responses to the infected cells. One is to inhibit CTL recognition by HIV-1 Nef-mediated down-regulation of MHC-I expression on the surface of infected cells. Here we report that Nef also reduces the expression of the non-classical MHC-I like CD1d molecule, a third lineage of antigen-presenting molecule, which presents lipid antigens. Nef achieves this by increasing internalization of CD1d molecules from the cell surface and retaining CD1d in the trans-Golgi-network (TGN). This effect depends on a tyrosine-based motif present in CD1 cytoplasmic tail as well as the actions of four Nef motifs, which are known to be involved in the down-regulation of MHC-I or CD4. These results suggest that Nef regulates intracellular trafficking of CD1d via a distinct but shared pathway with MHC-I and CD4. Thus, HIV-1 reduces the visibility of its infected cells not only to MHC-I-restricted T cells but also to CD1d-restricted NKT cells. Given that CD1d-restricted T cells have unique effector and regulatory functions in innate and adapted immune responses as compared with their counterpart MHC-restricted T cells, our data provide additional new insights into molecular basis of HIV-1-mediated damage to the immune system.

Willinger T, Freeman T, Herbert M, Hasegawa H, McMichael AJ, Callan MF. 2006. Human naive CD8 T cells down-regulate expression of the WNT pathway transcription factors lymphoid enhancer binding factor 1 and transcription factor 7 (T cell factor-1) following antigen encounter in vitro and in vivo. J Immunol, 176 (3), pp. 1439-1446. | Show Abstract

The transcription factors lymphoid enhancer binding factor 1 (LEF1) and transcription factor 7 (TCF7) (T cell factor-1 (TCF-1)) are downstream effectors of the WNT signaling pathway, which is a critical regulator of T cell development in the thymus. In this study, we show that LEF1 and TCF7 (TCF-1) are not only expressed in thymocytes, but also in mature T cells. Our data demonstrate that Ag encounter in vivo and engagement of the TCR or IL-15 receptor in vitro leads to the down-regulation of LEF1 and TCF7 (TCF-1) expression in human naive CD8 T cells. We further show that resting T cells preferentially express inhibitory LEF1 and TCF7 (TCF-1) isoforms and that T cell activation changes the isoform balance in favor of stimulatory TCF7 (TCF-1) isoforms. Altogether, our study suggests that proteins involved in the WNT signaling pathway not only regulate T cell development, but also peripheral T cell differentiation.

Cebere I, Dorrell L, McShane H, Simmons A, McCormack S, Schmidt C, Smith C, Brooks M et al. 2006. Phase I clinical trial safety of DNA- and modified virus Ankara-vectored human immunodeficiency virus type 1 (HIV-1) vaccines administered alone and in a prime-boost regime to healthy HIV-1-uninfected volunteers. Vaccine, 24 (4), pp. 417-425. | Show Abstract | Read more

DNA- and modified virus Ankara (MVA)-vectored candidate vaccines expressing human immunodeficiency virus type 1 (HIV-1) clade A-derived p24/p17 gag fused to a string of HLA class I epitopes, called HIVA, were tested in phase I trials in healthy, HIV-1/2-uninfected adults in Oxford, United Kingdom. Eighteen volunteers were vaccinated with pTHr.HIVA DNA (IAVI-001) alone, 8 volunteers received MVA.HIVA (IAVI-003) alone and 9 volunteers from study IAVI-001 were boosted with MVA.HIVA 9-14 months after DNA priming (IAVI-005). Immunogenicity results observed in these trials was published previously [Mwau M, Cebere I, Sutton J, Chikoti P, Winstone N, Wee EG-T, et al. An HIV-1 clade A vaccine in clinical trials: stimulation of HIV-specific T cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol 2004;85:911-9]. Here, we report on the safety of the two vaccines and the vaccine regimes. Overall, both candidate vaccines were safe and well tolerated. There were no reported vaccine-related adverse events over the 6-month period of the study and up to 2 years after the last vaccination. There were no moderate or severe local symptoms recorded after the pTHr.HIVA DNA intramuscular administration. Almost all participants experienced local reactogenicity events such as redness and induration after MVA.HIVA intradermal injection. Thus, the results from these initial small phase I trials administering the pTHr.HIVA DNA and MVA.HIVA vaccines either alone or in a prime-boost regime to healthy HIV-1/2-negative adults indicated that the vaccines were safe and warranted further testing of this approach in larger phase I/II studies.

McMichael AJ. 2006. HIV vaccines. Annu Rev Immunol, 24 (1), pp. 227-255. | Show Abstract | Read more

A prophylactic vaccine for HIV-1 is badly needed. Despite 20 years of effort, it is still a long way off. However, considerable progress has been made in understanding the problem. The virus envelope has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope. Anti-HIV-1 T cell responses are evaded by continuous mutation of the virus. Vaccine strategies that concentrate on stimulating T cell immunity will at best generate broadly reactive and persisting T cell responses that can suppress virus without preventing infection, limiting or preventing the damage the virus causes. The SIV macaque models give encouragement that this is possible, but they need further understanding. Therapeutic vaccination should also be considered.

Simmons A, Gangadharan B, Hodges A, Sharrocks K, Prabhakar S, García A, Dwek R, Zitzmann N, McMichael A. 2005. Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to positively regulate signaling. Immunity, 23 (6), pp. 621-634. | Show Abstract | Read more

Lentiviral Nef increases T cell signaling activity, but the molecular nature of the stimulus involved is incompletely described. We explored CD4 T cell lipid raft composition in the presence and absence of Nef. Here, the E2 ubiquitin-conjugating enzyme UbcH7, which acts in conjunction with c-Cbl, is absent from lipid rafts. This Nef-mediated exclusion is associated with failure of ubiquitination of activated Vav. In the presence of Nef, lipid raft Cdc42 is activated and forms a ternary complex between the c-Cbl-interacting protein p85Cool-1/betaPix and c-Cbl, displacing UbcH7 from rafts. Suppression of p85Cool-1/betaPix expression restores UbcH7 raft localization and Vav ubiquitination and diminishes Cdc42 activity. Moreover, p85Cool-1/betaPix knockdown attenuates HIV replication. Thresholds for activation of signaling involve the intricate balance of positive and negative regulators. Here we provide evidence for Nef disruption of a negative regulator of T cell signaling in promoting HIV replication.

Estcourt MJ, McMichael AJ, Hanke T. 2005. Altered primary CD8+ T cell response to a modified virus Ankara(MVA)-vectored vaccine in the absence of CD4+ T cell help. Eur J Immunol, 35 (12), pp. 3460-3467. | Show Abstract | Read more

T cell receptor-transgenic F5 mice were used to assess primary CD8+ T cell responses to a modified virus Ankara (MVA)-vectored vaccine in the absence of CD4+ T cell help. Naive, CD8-enriched, CFSE-labelled F5 cells were transferred into normal or CD4+ cell-depleted mice and the mice were vaccinated with MVA.HIVA-NP. At different time points during the primary response, F5 cells were re-isolated and analysed on divisional basis for a number of parameters. We demonstrated that the primary CD8+ T cell response in the absence of CD4+ T cell help differed from that in normal CD4+ cell-undepleted mice. While in the absence of CD4+ T cell help, the initial migratory progress from the local response to a systemic one was not grossly affected, the proportion of dying F5 cells during the expansion phase was markedly increased and resulted in an overall smaller expansion and significantly decreased frequency of CD8+ T cell memory after contraction. T cells primed without help displayed accelerated proliferation and activation, while expression of interferon-gamma remained similar. These phenomena were observed in the lymph nodes draining the MVA.HIVA-NP immunization site and were similar, but delayed by 2-3 days in spleen and non-draining lymph nodes.

Ranasinghe SR, Abidi SH, Rowland-Jones SL, McMichael AJ, Dong T, Davis SJ. 2005. Functional analysis of candidate CD8+T cell-derived anti-HIV factors IMMUNOLOGY, 116 pp. 80-80.

Larke N, Murphy A, Wirblich C, Teoh D, Estcourt MJ, McMichael AJ, Roy P, Hanke T. 2005. Induction of human immunodeficiency virus type 1-specific T cells by a bluetongue virus tubule-vectored vaccine prime-recombinant modified virus Ankara boost regimen. J Virol, 79 (23), pp. 14822-14833. | Show Abstract | Read more

In the absence of strategies for reliable induction of antibodies broadly neutralizing human immunodeficiency virus type 1 (HIV-1), vaccine efforts have shifted toward the induction of cell-mediated immunity. Here we describe the construction and immunogenicity of novel T-cell vaccine NS1.HIVA, which delivers the HIV-1 clade A consensus-derived immunogen HIVA on the surface of tubular structures spontaneously formed by protein NS1 of bluetongue virus. We demonstrated that NS1 tubules can accommodate a protein as large as 527 amino acids without losing their self-assembly capability. When injected into BALB/c mice by several routes, chimeric NS1.HIVA tubules induced HIV-1-specific major histocompatibility complex class I-restricted T cells. These could be boosted by modified virus Ankara expressing the same immunogen and generate a memory capable of gamma interferon (IFN-gamma) production, proliferation, and lysis of sensitized target cells. Induced memory T cells readily produced IFN-gamma 230 days postimmunization, and upon a surrogate virus challenge, NS1.HIVA vaccine alone decreased the vaccinia virus vv.HIVA load in ovaries by 2 orders of magnitude 280 days after immunization. Thus, because of its T-cell immunogenicity and antigenic simplicity, the NS1 delivery system could serve as a priming agent for heterologous prime-boost vaccination regimens. Its usefulness in primates, including humans, remains to be determined.

Amyes E, McMichael AJ, Callan MF. 2005. Human CD4+ T cells are predominantly distributed among six phenotypically and functionally distinct subsets. J Immunol, 175 (9), pp. 5765-5773. | Show Abstract

Human T cells are heterogeneous, varying in terms of their phenotype, functional capabilities, and history of Ag encounter. The derivation of a functionally relevant model for classifying CD4+ T cells has been hampered by limitations on the numbers of parameters that may be measured using classical four-color flow cytometry. In this study we have taken advantage of the introduction of reagents for five-color flow cytometry to develop a detailed, functionally meaningful scheme for classifying human CD4+ T cells. We show that CD4+ T cells are predominantly distributed among six of eight possible compartments, identified by the expression of CCR7, CD45RA, and CD28. We demonstrate novel phenotypic and functional correlates that justify the choice of these three molecules to define CD4+ T cell compartments. We note that CD4+ T cells with different Ag specificities are distributed differently among the six described subsets. On the basis of these results, we propose a cross-sectional model for classification of peripheral CD4+ T cells. Knowledge of where T cells lie on this model informs about their functional capacity and can reflect their history of Ag exposure.

Willinger T, Freeman T, Hasegawa H, McMichael AJ, Callan MF. 2005. Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets. J Immunol, 175 (9), pp. 5895-5903. | Show Abstract

Memory T cells are heterogeneous in terms of their phenotype and functional properties. We investigated the molecular profiles of human CD8 naive central memory (T(CM)), effector memory (T(EM)), and effector memory RA (T(EMRA)) T cells using gene expression microarrays and phospho-protein-specific intracellular flow cytometry. We demonstrate that T(CM) have a gene expression and cytokine signaling signature that lies between that of naive and T(EM) or T(EMRA) cells, whereas T(EM) and T(EMRA) are closely related. Our data define the molecular basis for the different functional properties of central and effector memory subsets. We show that T(EM) and T(EMRA) cells strongly express genes with known importance in CD8 T cell effector function. In contrast, T(CM) are characterized by high basal and cytokine-induced STAT5 phosphorylation, reflecting their capacity for self-renewal. Altogether, our results distinguish T(CM) and T(EM)/T(EMRA) at the molecular level and are consistent with the concept that T(CM) represent memory stem cells.

Estcourt MJ, Létourneau S, McMichael AJ, Hanke T. 2005. Vaccine route, dose and type of delivery vector determine patterns of primary CD8+ T cell responses. Eur J Immunol, 35 (9), pp. 2532-2540. | Show Abstract | Read more

The dynamics of primary CD8+ T cell responses following administration of modified virus Ankara (MVA)- and DNA-vectored vaccines was investigated in a mouse model. To overcome the low frequency of naive antigen-specific precursors and follow the early expansion events, naive CFSE-labelled T cell receptor-transgenic F5 lymphocytes were transferred into syngeneic non-transgenic recipients prior to vaccination. Using the i.d., i.v. and i.m. routes and increasing recombinant MVA (rMVA) vaccine doses, the primary response was analysed on a divisional basis at local and distant lymphoid organs at various times after vaccination. The results indicated that F5 cell divisions were initiated in the local draining lymph nodes and cells only after five to six divisions appeared at more distant sites. The rMVA dose affected frequencies of cells entering division and at the peak response. When priming induced by rMVA and plasmid DNA was compared, dramatic differences in the cycling patterns were observed with plasmid DNA inducing a response slower and more sustained over the first 2 wk than rMVA. Both rMVA and DNA induced comparable IFN-gamma production, which increased with cell divisions. Taken together, the vaccine type, dose and route have a strong influence on the spatial and temporal patterns of initial T cell responses.

Stewart-Jones GB, Gillespie G, Overton IM, Kaul R, Roche P, McMichael AJ, Rowland-Jones S, Jones EY. 2005. Structures of three HIV-1 HLA-B*5703-peptide complexes and identification of related HLAs potentially associated with long-term nonprogression. J Immunol, 175 (4), pp. 2459-2468. | Show Abstract

Long-term nonprogression during acute HIV infection has been strongly associated with HLA-B*5701 or HLA-B*5703. In this study, we present the high resolution crystal structures of HLA-B*5703 complexes with three HIV-1 epitopes: ISPRTLNAW (ISP), KAFSPEVIPMF (KAF-11), and KAFSPEVI (KAF-8). These reveal peptide anchoring at position 2 and their C termini. The different peptide lengths and primary sequences are accommodated by variation in the specific contacts made to the HLA-B*5703, flexibility in water structure, and conformational adjustment of side chains within the peptide-binding groove. The peptides adopt markedly different conformations, and trap variable numbers of water molecules, near a cluster of tyrosine side chains located in the central region of the peptide-binding groove. The KAF-11 epitope completely encompasses the shorter KAF-8 epitope but the peptides are presented in different conformations; the KAF-11 peptide arches out of the peptide-binding groove, exposing a significant main chain surface area. Bioinformatic analysis of the MHC side chains observed to contribute to the peptide anchor specificity, and other specific peptide contacts, reveals HLA alleles associated with long-term nonprogression and a number of related HLA alleles that may share overlapping peptide repertoires with HLA-B*5703 and thus may display a similar capacity for efficient immune control of HIV-1 infection.

Dorrell L, Yang H, Iversen AK, Conlon C, Suttill A, Lancaster M, Dong T, Cebere I et al. 2005. Therapeutic immunization of highly active antiretroviral therapy-treated HIV-1-infected patients: safety and immunogenicity of an HIV-1 gag/poly-epitope DNA vaccine. AIDS, 19 (12), pp. 1321-1323. | Show Abstract | Read more

In view of the global emergency posed by lack of access to highly active antiretroviral therapy (HAART) and the limitations of current drug regimens, alternative therapeutic strategies are urgently needed. Cellular immune responses elicited by HIV-1 exert some control over virus replication, therefore the enhancement of HIV-1-specific responses by therapeutic vaccination might lead to viral containment without HAART. We evaluated the safety and immunogenicity, in HIV-1-infected individuals under HAART suppression, of a DNA vaccine, pTHr.HIVA.

Iversen AKN, Learn GH, Skinhoj P, Mullins JI, McMichael AJ, Rambaut A. 2005. Preferential detection of HIV subtype C ' over subtype A in cervical cells from a dually infected woman AIDS, 19 (9), pp. 990-993. | Read more

Kaufmann SH, McMichael AJ. 2005. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat Med, 11 (4 Suppl), pp. S33-S44. | Show Abstract | Read more

Human immunodeficiency virus (HIV) and Mycobacterium tuberculosis annually cause 3 million and 2 million deaths, respectively. Last year, 600,000 individuals, doubly infected with HIV and M. tuberculosis, died. Since World War I, approximately 150 million people have succumbed to these two infections--more total deaths than in all wars in the last 2,000 years. Although the perceived threats of new infections such as SARS, new variant Creutzfeldt-Jakob disease and anthrax are real, these outbreaks have caused less than 1,000 deaths globally, a death toll AIDS and tuberculosis exact every 2 h. In 2003, 40 million people were infected with HIV, 2 billion with M. tuberculosis, and 15 million with both. Last year, 5 million and 50 million were newly infected with HIV or M. tuberculosis, respectively, with 2 million new double infections. Better control measures are urgently needed.

Ho LP, Merlin F, Gaber K, Davies RJ, McMichael AJ, Hugot JP. 2005. CARD 15 gene mutations in sarcoidosis. Thorax, 60 (4), pp. 354-355. | Read more

Ho LP, Urban BC, Thickett DR, Davies RJ, McMichael AJ. 2005. Deficiency of a subset of T-cells with immunoregulatory properties in sarcoidosis. Lancet, 365 (9464), pp. 1062-1072. | Show Abstract | Read more

BACKGROUND: Sarcoidosis is a multisystem disorder that predominantly involves the lungs, characterised by a T-helper 1 (Th1) biased CD4-positive T-cell response and granuloma formation, for which the explanation is unknown. A newly identified subset of T-cells with immunoregulatory functions, CD1d-restricted natural-killer T (NKT) cells, has been shown to protect against disorders with increased CD4-positive Th1 responses in animals. We explored whether abnormalities in these cells are implicated in the pathogenesis of sarcoidosis. METHODS: We generated fluorescence-labelled CD1d-tetrameric complexes and used them, with monoclonal antibodies to Valpha24 and Vbeta11 T-cell receptor, to assess the frequency of CD1d-restricted NKT cells in the peripheral blood of 60 patients with histologically proven sarcoidosis (16 with Lofgren's syndrome) and 60 healthy controls. Lung lymphocytes were also analysed in 16 of the patients with sarcoidosis. FINDINGS: CD1d-restricted NKT cells were absent or greatly reduced in peripheral blood from all patients with sarcoidosis, except those with Lofgren's syndrome (median proportion of lymphocytes 0.01% [IQR 0-0.03] vs 0.06% [0.03-0.12] in controls; p=0.0004). The deficiency was found in both acute and resolved disease and was unrelated to systemic corticosteroid therapy. There was no difference in the proportion of CD1d-restricted NKT cells between peripheral blood and lungs in patients, suggesting that the peripheral-blood deficiency is not due to sequestration of these cells in the lungs. The NKT cells were not observed in mediastinal lymph nodes or granulomatous lesions. CD1d expression on antigen-presenting cells of patients was normal, thus the deficiency of CD1d-restricted NKT cells is not explained by abnormal CD1d expression. INTERPRETATION: Loss of immunoregulation by CD1d-restricted NKT cells could explain the amplified and persistent T-cell activity that characterises sarcoidosis. RELEVANCE TO PRACTICE: Our findings give new insight into the pathogenesis of sarcoidosis and draw attention to a potential target for therapeutic modulation in sarcoidosis.

Hanke T, McMichael AJ, Dennis MJ, Sharpe SA, Powell LA, McLoughlin L, Crome SJ. 2005. Biodistribution and persistence of an MVA-vectored candidate HIV vaccine in SIV-infected rhesus macaques and SCID mice. Vaccine, 23 (12), pp. 1507-1514. | Show Abstract | Read more

Recombinant modified vaccinia virus Ankara (MVA) is together with a few other attenuated viral vectors on the forefront of human immunodeficiency virus type 1 (HIV-1) vaccine development. As such, MVA-vectored vaccines are likely to be administered into immunocompromized individuals. Here, we demonstrated in a good laboratory practice study safety and biological clearance of candidate HIV-1 vaccine MVA.HIVA in simian immunodeficiency virus (SIV)-infected rhesus macaques and mice with a severe combined immunodeficiency (SCID) following an intradermal vaccine administration. In SIV-infected macaques, MVA.HIVA DNA was undetectable by nested PCR 6 weeks after dosing. In SCID mice, the MVA.HIVA vaccine was well tolerated and a positive PCR signal was only observed at the site of injection 49 days after dosing in four out of six mice, but even these sites were negative by day 81 post-injection. Therefore, the MVA.HIVA vaccine is considered safe for application in phase I clinical trials in HIV-1-infected human subjects. These results also contribute to the confidence of using MVA as a smallpox vaccine.

Stewart-Jones GB, di Gleria K, Kollnberger S, McMichael AJ, Jones EY, Bowness P. 2005. Crystal structures and KIR3DL1 recognition of three immunodominant viral peptides complexed to HLA-B*2705. Eur J Immunol, 35 (2), pp. 341-351. | Show Abstract | Read more

We have solved the crystal structures of three HLA-B*2705-peptide complexes with the immunodominant viral peptides: EBV EBNA3C 258-266 (RRIYDLIEL), influenza (flu) nucleoprotein NP383-391 (SRYWAIRTR), and HIV gag 264-273 (KRWIILGLNK). Long-term non-progression during HIV infection has been associated with presentation by HLA-B*2705, and T cell recognition, of the highly immunodominant KRWIILGLNK peptide. The tight hydrogen-bonding network observed between the HLA-B*2705 B-pocket and the peptide P2 arginine guanadinium anchor explains why mutation of this residue during HIV infection results in loss of peptide binding, immune escape and progression to AIDS. Prominent, solvent-exposed structures within these peptides may participate in generating T cell responses to these immunodominant epitopes. In the HLA-B*2705 complex with flu NP383-391, the amino acid side chains of residues 4, 7 and 8 are solvent-exposed whilst in the HIV decamer, the main-chain bulges into the solvent around P7. Thus, HLA-B*2705 presents viral peptides in a range of conformations. Tetrameric complexes of HLA-B*2705 with the HIV and flu but not EBV peptides bound strongly to the killer-Ig-like receptor (KIR)3DL1. Substitution of EBV P8 glutamate to threonine allowed recognition by KIR3DL1. In the HLA-B*2705-EBV structure the P8 glutamate side chain is solvent-exposed and may inhibit KIR3DL1 binding through electrostatic forces.

Poon K, Montamat-Sicotte D, Cumberbatch N, McMichael AJ, Callan MF. 2005. Expression of leukocyte immunoglobulin-like receptors and natural killer receptors on virus-specific CD8+ T cells during the evolution of Epstein-Barr virus-specific immune responses in vivo. Viral Immunol, 18 (3), pp. 513-522. | Show Abstract | Read more

Antigen-primed cytotoxic T lymphocytes (CTL) may express leukocyte immunoglobulin-like receptors (LILRs) and natural killer receptors (NKRs). Published work suggests that expression of some of these receptors confers survival advantage, leading to the idea that cells expressing such receptors may accumulate as an antigen-specific response evolves. Here we tested this hypothesis by analyzing expression of CD85j (also known as LILRB1 or ILT2), KIRs, CD94, and CD161 by Epstein- Barr virus (EBV)-specific CTL during the primary and persistent phases of EBV infection in humans. During primary infection, few EBV-specific CTL expressed these receptors and this proportion was equally low in early persistent infection. Thus, expression of these molecules does not influence capacity to survive downregulation of the primary response. However, in donors persistently infected with EBV for many years, a significantly higher proportion of EBV-specific CTL expressed CD85j and NKRs, suggesting that cells expressing these receptors can accumulate with time. Using FACS analysis, we confirmed, at a single cell level, that expression of CD85j, defined by staining with the antibody VMP55, was associated with reduced capacity of EBV-specific CD8+ T cells to respond to antigen. Thus, in the later stages of persistent infection, protective immunity to EBV may be reduced due to the preferential accumulation of hyporesponsive EBV-specific CD8+ T cells.

Dong T, Stewart-Jones G, Chen N, Easterbrook P, Xu X, Papagno L, Appay V, Weekes M et al. 2004. HIV-specific cytotoxic T cells from long-term survivors select a unique T cell receptor. J Exp Med, 200 (12), pp. 1547-1557. | Show Abstract | Read more

HIV-specific cytotoxic T lymphocytes (CTL) are important in controlling HIV replication, but the magnitude of the CTL response does not predict clinical outcome. In four donors with delayed disease progression we identified Vbeta13.2 T cell receptors (TCRs) with very similar and unusually long beta-chain complementarity determining region 3 (CDR3) regions in CTL specific for the immunodominant human histocompatibility leukocyte antigens (HLA)-B8-restricted human immunodeficiency virus-1 (HIV-1) nef epitope, FLKEKGGL (FL8). CTL expressing Vbeta13.2 TCRs tolerate naturally arising viral variants in the FL8 epitope that escape recognition by other CTL. In addition, they expand efficiently in vitro and are resistant to apoptosis, in contrast to FL8-specific CTL using other TCRs. Selection of Vbeta13.2 TCRs by some patients early in the FL8-specific CTL response may be linked with better clinical outcome.

Lee JK, Stewart-Jones G, Dong T, Harlos K, Di Gleria K, Dorrell L, Douek DC, van der Merwe PA, Jones EY, McMichael AJ. 2004. T cell cross-reactivity and conformational changes during TCR engagement. J Exp Med, 200 (11), pp. 1455-1466. | Show Abstract | Read more

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2-restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR-peptide-major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR-peptide-MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.

Hambleton S, Valeyev NV, Muranyi A, Knott V, Werner JM, McMichael AJ, Handford PA, Downing AK. 2004. Structural and functional properties of the human notch-1 ligand binding region. Structure, 12 (12), pp. 2173-2183. | Show Abstract | Read more

We present NMR structural and dynamics analysis of the putative ligand binding region of human Notch-1, comprising EGF-like domains 11-13. Functional integrity of an unglycosylated, recombinant fragment was confirmed by calcium-dependent binding of tetrameric complexes to ligand-expressing cells. EGF modules 11 and 12 adopt a well-defined, rod-like orientation rigidified by calcium. The interdomain tilt is similar to that found in previously studied calcium binding EGF pairs, but the angle of twist is significantly different. This leads to an extended double-stranded beta sheet structure, spanning the two EGF modules. Based on the conservation of residues involved in interdomain hydrophobic packing, we propose this arrangement to be prototypical of a distinct class of EGF linkages. On this premise, we have constructed a model of the 36 EGF modules of the Notch extracellular domain that enables predictions to be made about the general role of calcium binding to this region.

Dalbeth N, Gundle R, Davies RJ, Lee YC, McMichael AJ, Callan MF. 2004. CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation. J Immunol, 173 (10), pp. 6418-6426. | Show Abstract

Human NK cells may be divided into a CD56(dim) subset and a CD56(bright) subset. In peripheral blood, CD56(dim) NK cells dominate, whereas in lymph nodes, CD56(bright) NK cells are more common. In this study we show that CD56(bright) NK cells accumulate within inflammatory lesions in a wide variety of clinical diseases affecting several different anatomical sites. We demonstrate that when activated by the monokines IL-12, IL-15, and IL-18, these NK cells promote TNF-alpha production by CD14(+) monocytes in a manner that is dependent on cell:cell contact. Conversely, CD14(+) monocytes synergize with monokines to promote IFN-gamma production by these NK cells. Again, this interaction is dependent on cell:cell contact. The experiments show that CD56(bright) NK cells accumulate in inflammatory lesions and, in the appropriate cytokine environment, can engage with CD14(+) monocytes in a reciprocal activatory fashion, thereby amplifying the inflammatory response. Such a positive feedback loop is likely to be important in the pathogenesis of chronic inflammatory conditions such as rheumatoid arthritis.

Kaul R, Rutherford J, Rowland-Jones SL, Kimani J, Onyango JI, Fowke K, MacDonald K, Bwayo JJ, McMichael AJ, Plummer FA. 2004. HIV-1 Env-specific cytotoxic T-lymphocyte responses in exposed, uninfected Kenyan sex workers: a prospective analysis. AIDS, 18 (15), pp. 2087-2089. | Show Abstract | Read more

The prospective significance of HIV-specific cytotoxic T lymphocyte (CTL) responses in highly exposed, persistently seronegative populations is unknown. In 1996-1997 we screened for CTL responses against HIV clade B Env in 39 recently enrolled Kenyan female sex workers, and followed these women prospectively. Annual HIV incidence was 5.8%. CTL were independently associated with age and recent HIV-1 exposure,but were not prospectively associated with protection in a multivariable model that included HIV-1 exposure and duration of sex work.

Kollnberger S, Bird LA, Roddis M, Hacquard-Bouder C, Kubagawa H, Bodmer HC, Breban M, McMichael AJ, Bowness P. 2004. HLA-B27 heavy chain homodimers are expressed in HLA-B27 transgenic rodent models of spondyloarthritis and are ligands for paired Ig-like receptors. J Immunol, 173 (3), pp. 1699-1710. | Show Abstract

HLA-B27 transgenic rats and strains of HLA-B27-transgenic beta(2)-microglobulin (beta(2)m)-deficient mice develop a multisystem inflammatory disease affecting the joints, skin, and bowel with strong similarity to human spondyloarthritis. We show that HLA-B27 transgenic mice and rats express HC10-reactive, beta(2)m-free HLA-B27 homodimers (B27(2)) and multimers, both intracellularly and at the cell surface of leukocytes, including rat dendritic cells. Fluorescent-labeled tetrameric complexes of HLA-B27 homodimers (B27(2) tetramers) bind to populations of lymphocytes, monocytes, and dendritic cells. The murine (and probably rat) paired Ig-like receptors (PIRs) are ligands for B27(2). Thus, B27(2) tetramers stain RBL cells transfected with murine activating PIR-A4 and inhibitory PIR-B receptors. Murine PIR-A and -B can be immunoprecipitated from the RAW264.7 macrophage cell line, and murine PIR-A can be immunoprecipitated from the J774.A1 line using B27(2). B27(2) tetramer staining corresponds to the distribution of PIR expression on lymphoid and myeloid cells and on murine macrophage cell lines. B27(2) can induce TNF-alpha release from the J774.A1 macrophage cell line. The binding of B27(2) to PIR is inhibited by HC10, an mAb that ameliorates arthritis in HLA-B27(+) beta(2)m(-/-) mice. The expression and PIR recognition of B27(2) could explain the pathogenesis of rodent spondyloarthritis.

Nkolola JP, Wee EG, Im EJ, Jewell CP, Chen N, Xu XN, McMichael AJ, Hanke T. 2004. Engineering RENTA, a DNA prime-MVA boost HIV vaccine tailored for Eastern and Central Africa. Gene Ther, 11 (13), pp. 1068-1080. | Show Abstract | Read more

For the development of human immunodeficiency virus type 1 (HIV-1) vaccines, traditional approaches inducing virus-neutralizing antibodies have so far failed. Thus the effort is now focused on elicitation of cellular immunity. We are currently testing in clinical trials in the United Kingdom and East Africa a T-cell vaccine consisting of HIV-1 clade A Gag-derived immunogen HIVA delivered in a prime-boost regimen by a DNA plasmid and modified vaccinia virus Ankara (MVA). Here, we describe engineering and preclinical development of a second immunogen RENTA, which will be used in combination with the present vaccine in a four-component DNA/HIVA-RENTA prime-MVA/HIVA-RENTA boost formulation. RENTA is a fusion protein derived from consensus HIV clade A sequences of Tat, reverse transcriptase, Nef and gp41. We inactivated the natural biological activities of the HIV components and confirmed immunogenicities of the pTHr.RENTA and MVA.RENTA vaccines in mice. Furthermore, we demonstrated in mice and rhesus monkeys broadening of HIVA-elicited T-cell responses by a parallel induction of HIVA- and RENTA-specific responses recognizing multiple HIV epitopes.

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.

Ho LP, Urban BC, Jones L, Ogg GS, McMichael AJ. 2004. CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion. J Immunol, 172 (12), pp. 7350-7358. | Show Abstract

Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.

Estcourt MJ, McMichael AJ, Hanke T. 2004. DNA vaccines against human immunodeficiency virus type 1. Immunol Rev, 199 (1), pp. 144-155. | Show Abstract | Read more

Development of a vaccine against human immunodeficiency virus type 1 (HIV-1) is the main hope for controlling the acquired immunodeficiency syndrome pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4+ helper T cells, and CD8+ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell-mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. This review focuses on the stimulation of HIV-specific T cells and discusses in the context of the current 'state-of-art' of DNA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development.

Mwau M, Cebere I, Sutton J, Chikoti P, Winstone N, Wee EG, Beattie T, Chen YH et al. 2004. A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol, 85 (Pt 4), pp. 911-919. | Show Abstract | Read more

The immunogenicities of candidate DNA- and modified vaccinia virus Ankara (MVA)-vectored human immunodeficiency virus (HIV) vaccines were evaluated on their own and in a prime-boost regimen in phase I clinical trials in healthy uninfected individuals in the United Kingdom. Given the current lack of approaches capable of inducing broad HIV-neutralizing antibodies, the pTHr.HIVA DNA and MVA.HIVA vaccines focus solely on the induction of cell-mediated immunity. The vaccines expressed a common immunogen, HIVA, which consists of consensus HIV-1 clade A Gag p24/p17 proteins fused to a string of clade A-derived epitopes recognized by cytotoxic T lymphocytes (CTLs). Volunteers' fresh peripheral blood mononuclear cells were tested for HIV-specific responses in a validated gamma interferon enzyme-linked immunospot (ELISPOT) assay using four overlapping peptide pools across the Gag domain and three pools of known CTL epitopes present in all of the HIVA protein. Both the DNA and the MVA vaccines alone and in a DNA prime-MVA boost combination were safe and induced HIV-specific responses in 14 out of 18, seven out of eight and eight out of nine volunteers, respectively. These results are very encouraging and justify further vaccine development.

Klausner RD, Fauci AS, Corey L, Nabel GJ, Gayle H, Berkley S, Haynes BF, Baltimore D et al. 2004. The challenges of an HIV vaccine enterprise - Response SCIENCE, 303 (5662), pp. 1296-1297.

Papagno L, Spina CA, Marchant A, Salio M, Rufer N, Little S, Dong T, Chesney G et al. 2004. Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection. PLoS Biol, 2 (2), pp. E20. | Show Abstract | Read more

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8(+) T-cells and the use of an in vitro model of naïve CD8(+) T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8(+) T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8(+) T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8(+) and CD4(+) T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.

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Dalbeth N, Gundle R, Davies RJO, Lee YCG, McMichael AJ, Callan MFC. 2004. CD56<sup>bright</sup> NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation Journal of Immunology, 173 (10), pp. 6418-6426. | Show Abstract

Human NK cells may be divided into a CD56dim subset and a CD56bright subset. In peripheral blood, CD56dim NK cells dominate, whereas in lymph nodes, CD56bright NK cells are more common. In this study we show that CD56bright NK cells accumulate within inflammatory lesions in a wide variety of clinical diseases affecting several different anatomical sites. We demonstrate that when activated by the monokines IL-12, IL-15, and IL-18, these NK cells promote TNF-α production by CD14+ monocytes in a manner that is dependent on cell:cell contact Conversely, CD14+ monocytes synergize with monokines to promote IFN-γ production by these NK cells. Again, this interaction is dependent on cell:celll contact The experiments show that CD56bright NK cells accumulate in inflammatory lesions and, in the appropriate cytokine environment, can engage with CD14+ monocytes in a reciprocal activatory fashion, thereby amplifying the inflammatory response. Such a positive feedback loop is likely to be important in the pathogenesis of chronic inflammatory conditions such as rheumatoid arthritis.

Roddis M, Carter RW, Sun MY, Weissensteiner T, McMichael AJ, Bowness P, Bodmer HC. 2004. Fully functional HLA B27-restricted CD4+ as well as CD8+ T cell responses in TCR transgenic mice. J Immunol, 172 (1), pp. 155-161. | Show Abstract

The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.

Zinkernagel RM, Wolf H, Klasner RD, Fauci AS, Corey L, Nabel GJ, Gayle H, Berkley S et al. 2004. The Challenges of an HIV Vaccine Enterprise [2] (multiple letters) Science, 303 (5662), pp. 1294-1297. | Read more

Amyes E, Hatton C, Montamat-Sicotte D, Gudgeon N, Rickinson AB, McMichael AJ, Callan MF. 2003. Characterization of the CD4+ T cell response to Epstein-Barr virus during primary and persistent infection. J Exp Med, 198 (6), pp. 903-911. | Show Abstract | Read more

The CD8+ T cell response to Epstein-Barr virus (EBV) is well characterized. Much less is known about the evolution of the CD4+ T cell response. Here we show that EBV stimulates a primary burst of effector CD4+ T cells and this is followed by a period of down-regulation. A small population of EBV-specific effector CD4+ T cells survives during the lifelong persistent phase of infection. The EBV-specific effector CD4+ T cells accumulate within a CD27+ CD28+ differentiation compartment during primary infection and remain enriched within this compartment throughout the persistent phase of infection. Analysis of CD4+ T cell responses to individual epitopes from EBV latent and lytic cycle proteins confirms the observation that the majority of the effector cells express both CD27 and CD28, although CD4+ T cells specific for lytic cycle antigens have a greater tendency to express CD45RA than those specific for the latent antigens. In clear contrast, effector CD4+ T cells specific for cytomegalovirus (CMV) accumulate within the CD27- CD28+ and CD27- CD28- compartments. There are striking parallels in terms of the differentiation of CD8+ T cells specific for EBV and CMV. The results challenge current ideas on the definition of memory subsets.

Bland FA, Lemberg MK, McMichael AJ, Martoglio B, Braud VM. 2003. Requirement of the proteasome for the trimming of signal peptide-derived epitopes presented by the nonclassical major histocompatibility complex class I molecule HLA-E. J Biol Chem, 278 (36), pp. 33747-33752. | Show Abstract | Read more

The nonclassical major histocompatibility complex class I molecule HLA-E acts as a ligand for CD94/NKG2 receptors on the surface of natural killer cells and a subset of T cells. HLA-E presents closely related nonameric peptide epitopes derived from the highly conserved signal sequences of classical major histocompatibility complex class I molecules as well as HLA-G. Their generation requires cleavage of the signal sequence by signal peptidase followed by the intramembrane-cleaving aspartic protease, signal peptide peptidase. In this study, we have assessed the subsequent proteolytic requirements leading to generation of the nonameric HLA-E peptide epitopes. We show that proteasome activity is required for further processing of the peptide generated by signal peptide peptidase. This constitutes the first example of capture of a naturally derived short peptide by the proteasome, producing a class I peptide ligand.

Whelan KT, Lin CL, Cella M, McMichael AJ, Austyn JM, Rowland-Jones SL. 2003. The HIV protease inhibitor indinavir reduces immature dendritic cell transendothelial migration. Eur J Immunol, 33 (9), pp. 2520-2530. | Show Abstract | Read more

Indinavir (IDV) is a protease inhibitor that successfully suppresses HIV-1 replication as part of anti-retroviral therapy. There is evidence to suggest that IDV may also act non-specifically upon host proteases. In this study we investigated whether IDV could modulate protease-dependent molecules involved in dendritic cell (DC) migration - a pivotal process in immunoregulation. Human monocyte-derived DC were exposed to IDV (IDV-DC) and transendothelial migration (TEM) to inflammatory chemokines was determined. TEM of IDV-DC was significantly impaired compared to non-treated DC (p<0.01). Phenotypic analysis revealed that IDV-DC had reduced DC-SIGN expression, correlating with reduced adhesion to immobilized ICAM-2. Nevertheless, the reduction in migration following exposure to IDV could not be fully attributable to DC-SIGN interactions alone. Investigation of IDV-DC interactions with the underlying matrix protein, fibronectin, demonstrated that IDV significantly impaired DC binding to immobilized fibronectin (p<0.01). IDV appeared to act upon VLA-4 and VLA-5 since addition of antagonist monoclonal antibodies (mAb) similarly reduced adhesion of non-treated DC to fibronectin. Combined blockade of DC using anti-VLA-4, VLA-5 and anti-DC-SIGN mAb inhibited TEM to a similar extent as IDV. Our results strongly suggest that IDV inhibits host proteases necessary for DC migration and may, therefore, affect DC immunoregulation in HIV-1-infected patients.

Evans EJ, Hene L, Sparks LM, Dong T, Retiere C, Fennelly JA, Manso-Sancho R, Powell J et al. 2003. The T cell surface--how well do we know it? Immunity, 19 (2), pp. 213-223. | Show Abstract | Read more

The overall degree of complexity of the T cell surface has been unclear, constraining our understanding of its biology. Using global gene expression analysis, we show that 111 of 374 genes encoding well-characterized leukocyte surface antigens are expressed by a resting cytotoxic T cell. Unexpectedly, of 97 stringently defined, T cell-specific transcripts with unknown functions that we identify, none encode proteins with the modular architecture characteristic of 80% of leukocyte surface antigens. Only two encode proteins with membrane topologies found exclusively in cell surface molecules. Our analysis indicates that the cell type-specific composition of the resting CD8+ T cell surface is now largely defined, providing an insight into the overall compositional complexity of the mammalian cell surface and a framework for formulating systematic models of T cell surface-dependent processes, such as T cell receptor triggering.

Stewart-Jones GB, McMichael AJ, Bell JI, Stuart DI, Jones EY. 2003. A structural basis for immunodominant human T cell receptor recognition. Nat Immunol, 4 (7), pp. 657-663. | Show Abstract | Read more

The anti-influenza CD8+ T cell response in HLA-A2-positive adults is almost exclusively directed at residues 58-66 of the virus matrix protein (MP(58-66)). V(beta)17V(alpha)10.2 T cell receptors (TCRs) containing a conserved arginine-serine-serine sequence in complementarity determining region 3 (CDR3) of the V(beta) segment dominate this response. To investigate the molecular basis of immunodominant selection in an outbred population, we have determined the crystal structure of V(beta)17V(alpha)10.2 in complex with MP(58-66)-HLA-A2 at a resolution of 1.4 A. We show that, whereas the TCR typically fits over an exposed side chain of the peptide, in this structure MP(58-66) exposes only main chain atoms. This distinctive orientation of V(beta)17V(alpha)10.2, which is almost orthogonal to the peptide-binding groove of HLA-A2, facilitates insertion of the conserved arginine in V(beta) CDR3 into a notch in the surface of MP(58-66)-HLA-A2. This previously unknown binding mode underlies the immunodominant T cell response.

McMichael AJ, Hanke T. 2003. HIV vaccines 1983-2003. Nat Med, 9 (7), pp. 874-880. | Show Abstract | Read more

Twenty years after the discovery of HIV, there is still no vaccine. This year, an envelope vaccine aimed at stimulating neutralizing antibodies was unable to protect against infection in phase 3 trials. But more than 20 HIV vaccines designed to stimulate T-cell responses are being developed. Will any of them work?

Klausner RD, Fauci AS, Corey L, Nabel GJ, Gayle H, Berkley S, Haynes BF, Baltimore D et al. 2003. Medicine. The need for a global HIV vaccine enterprise. Science, 300 (5628), pp. 2036-2039. | Show Abstract | Read more

A new collaborative model of research is needed to increase resources, to prioritize the R (ii) to increase the pace, reduce the overlap, and more systematically explore the elements of and delivery systems for vaccines; (iii) to use common standards for the prompt comparative testing of vaccine candidates; (iv) to expand resources for manufacturing vaccine candidates to speed their use in human trials; and (v) to increase the capacity for international clinical trials and to focus this effort toward quickly measuring the effectiveness of vaccine protection as prototype vaccine candidates are identified.

Marchant A, Appay V, Van Der Sande M, Dulphy N, Liesnard C, Kidd M, Kaye S, Ojuola O et al. 2003. Mature CD8(+) T lymphocyte response to viral infection during fetal life. J Clin Invest, 111 (11), pp. 1747-1755. | Show Abstract | Read more

Immunization of newborns against viral infections may be hampered by ineffective CD8(+) T cell responses. To characterize the function of CD8(+) T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8(+) T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8(+) T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28(-)CD27(+)CD45RO(+), perforin(low)), and they acquired a late differentiation phenotype (CD28(-)CD27(-)CD45RA(+), perforin(high)) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8(+) T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

Bird LA, Peh CA, Kollnberger S, Elliott T, McMichael AJ, Bowness P. 2003. Lymphoblastoid cells express HLA-B27 homodimers both intracellularly and at the cell surface following endosomal recycling. Eur J Immunol, 33 (3), pp. 748-759. | Show Abstract | Read more

The MHC class I allele HLA-B27 is very strongly associated with development of autoimmune spondyloarthritis, although the disease mechanism remains unknown. Class I molecules classically associate in the endoplasmic reticulum (ER) with beta2-microglobulin (beta(2)m) and antigenic peptides for cell surface expression and presentation to T cells. We have previously shown that HLA-B27 is capable of forming beta(2)m-free disulfide-bonded homodimers in vitro. Here we show that HLA-B27 forms disulfide-bonded homodimers in vivo by two distinct pathways. HLA-B27 homodimers form in the ER but appear unable to egress to the cell surface in human cells. Cell surface HLA-B27 homodimers are abundantly expressed in a variety of lymphoid cell lines. Experiments with inhibitors indicate that HLA-B27 homodimers can arise from cell-surface heterodimers via an endosome-dependent recycling pathway. HLA-B27 homodimer expression on the cell surface of 721.220 is dependent on the unpaired cysteine(67) and is inhibited by restoration of tapasin function or by incubation with peptides that bind strongly to HLA-B27 heterodimers. Cell surface expressed HLA-B27 homodimers are likely to be immunologically reactive ligands for NK family immunoreceptors and, hence, could play a pathogenic role in spondyloarthritis.

Mwau M, McMichael AJ. 2003. A review of vaccines for HIV prevention. J Gene Med, 5 (1), pp. 3-10. | Show Abstract | Read more

HIV/AIDS has become the most devastating pandemic in recorded history. It has killed 40 million people in the last 20 years and the World Health Organisation estimated that at least 14,000 new infections occurred daily in 2001. There will be up to 100 million new infections in the next 10 years (for current updates, visit http://www.unaids.org/epidemic_update/). Most HIV infections occur in the developing world, and the adverse social and economic impact of the HIV/AIDS pandemic, particularly in the developing world, is unprecedented. Highly active antiretroviral therapy (HAART) has had significant effects on HIV/AIDS in the developed world. The drugs have acted to prolong survival, reduce the viral load, and to alleviate suffering. However, the incidence of side effects and resistance is high and the drugs are unaffordable and unavailable in the developing world. HAART regimens are difficult to comply with. Public health efforts to modify the behaviour, attitude and culture that accelerate the spread of HIV/AIDS have had only modest success. There is urgent need for a prophylactic and/or therapeutic HIV vaccine. This is a review of the obstacles and current trends in HIV vaccine development.

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.

McMichael AJ, Rowland-Jones SL. 2002. HIV: Bad news for stop--start therapy? Nature, 420 (6914), pp. 371-373. | Show Abstract | Read more

An HIV-infected patient who was being treated with anti-retroviral drugs in a 'stop-start' protocol has become infected with a second HIV strain, raising questions about both the treatment strategy and vaccine development.

Hanke T, McMichael AJ, Samuel RV, Powell LA, McLoughlin L, Crome SJ, Edlin A. 2002. Lack of toxicity and persistence in the mouse associated with administration of candidate DNA- and modified vaccinia virus Ankara (MVA)-based HIV vaccines for Kenya. Vaccine, 21 (1-2), pp. 108-114. | Show Abstract | Read more

Toxicity, biodistribution and persistence of candidate HIV vaccines pTHr.HIVA, a recombinant DNA, and MVA.HIVA, a recombinant modified vaccinia virus Ankara, were determined in the Balb/c mouse. The mice were injected with either two doses of intramuscular pTHr.HIVA DNA (50 microg each, separated by an interval of 14 days), two doses of intradermal MVA.HIVA (10(6) plaque-forming units each, separated by an interval of 14 days), or a combination of the two vaccines, each given in two doses, in a prime-boost regimen. The study showed no significant toxic effects, either local or systemic, under any of these employed dosing regimens. With the exception of the sites of delivery, the vaccine-derived HIVA DNA sequences were undetectable 5 weeks after the last dosing. Thus, both the vaccines alone and in a combination were considered safe and suitable for the use in phase I trials in humans.

Seneviratne SL, Jones L, King AS, Black A, Powell S, McMichael AJ, Ogg GS. 2002. Allergen-specific CD8(+) T cells and atopic disease. J Clin Invest, 110 (9), pp. 1283-1291. | Show Abstract | Read more

Considerable evidence suggests that IL-10 may have a role in the manifestation of atopic disease. We sought to test the hypothesis that at the single cell level, allergen-specific T cells have diminished IL-10 production capacity in severely affected atopics compared with asymptomatic atopics. We defined three A*0201-restricted Der p 1 CD8(+) T cell epitopes. Using human leukocyte antigen-A*0201-peptide (HLA-A*0201-peptide) tetrameric complexes and enzyme-linked immunospot assays to analyze peripheral blood mononuclear cells from A*0201-positive severely symptomatic atopics, asymptomatic atopics, and nonatopic controls, we observed a significant association between the frequency of the Der p 1-specific CD8(+) T cells and disease activity. The specific T cells expressed an antigen-experienced cell surface phenotype, and 45.7% were positive for cutaneous lymphocyte-associated antigen. The specific T cells were able to produce IFN-gamma efficiently, but their IL-10 production was significantly reduced in severely affected atopics. In contrast, viral-specific CD8(+) T cells were able to produce equivalent amounts of IL-10 in the severely affected atopics compared with asymptomatic atopics and nonatopics. Through defining the first human atopic allergen HLA class I epitopes, we have provided a possible cellular mechanism to link the previous association of low IL-10 levels and severe atopic disease. These data are consistent with a role for CD8(+) T cells in atopic disease pathogenesis and may provide a basis for future T cell immunotherapy strategies.

van Baarle D, Kostense S, Hovenkamp E, Ogg G, Nanlohy N, Callan MF, Dukers NH, McMichael AJ, van Oers MH, Miedema F. 2002. Lack of Epstein-Barr virus- and HIV-specific CD27- CD8+ T cells is associated with progression to viral disease in HIV-infection. AIDS, 16 (15), pp. 2001-2011. | Show Abstract | Read more

OBJECTIVE: Despite readily detectable virus-specific CD8+ T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein-Barr virus (EBV)-specific CD8+ T cells was performed. DESIGN: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV-associated non-Hodgkin lymphoma's (NHL), both number and phenotype of virus-specific CD8+ T cells was studied longitudinally. METHODS: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells. RESULTS: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27- T cells, in contrast to rising numbers of EBV-specific CD27- CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS-related NHL had very low numbers of EBV-specific CD27- CD8+ T cells. Higher numbers of HIV-specific CD27- CD8+ T cells were associated with delayed disease progression. Virus-specific CD27- T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function. CONCLUSIONS: Taken together, our data indicate that virus-specific CD27- T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27- effector T cells may lead to progression of viral disease.

Allen TM, Jing P, Calore B, Horton H, O'Connor DH, Hanke T, Piekarczyk M, Ruddersdorf R et al. 2002. Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection. J Virol, 76 (20), pp. 10507-10511. | Show Abstract | Read more

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.

Allan DS, Lepin EJ, Braud VM, O'Callaghan CA, McMichael AJ. 2002. Tetrameric complexes of HLA-E, HLA-F, and HLA-G. J Immunol Methods, 268 (1), pp. 43-50. | Show Abstract | Read more

HLA-E, HLA-F, and HLA-G are human nonclassical MHC class Ib molecules. To study the function and identify potential ligands of these molecules, we constructed tetrameric complexes. In this brief review, we discuss the methods used to produce such tetramers and the interesting results they provided. HLA-E tetramers bound to natural killer (NK) cells and T cells, allowing the identification of CD94/NKG2 molecules as receptors for HLA-E. HLA-G tetramers interacted with immunoglobulin-like transcript-2 (ILT2) and ILT4 receptors, aiding the understanding of HLA-G function during pregnancy. Tetrameric complexes of HLA-F also bound to ILT2 and ILT4.

Mwau M, McMichael AJ, Hanke T. 2002. Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Res Hum Retroviruses, 18 (9), pp. 611-618. | Show Abstract | Read more

The enzyme-linked immunosorbent (ELISPOT) assay, which enumerates peripheral blood mononuclear cells (PBMCs) releasing interferon gamma (IFN-gamma) on specific antigen stimulation, is becoming the assay of choice for evaluation of vaccine-induced cell-mediated immune responses in many clinical trials. A properly conducted trial requires the assays to be validated, especially should the trial lead to vaccine licensure. Here, the design and validation of an ELISPOT assay are described for use in clinical trials of candidate human immunodeficiency virus (HIV) vaccines, using a particular immunogen termed HIVA. This assay employs eight pools of 20 to 23 peptides each: seven pools are derived from the immunogen and one pool is derived from cytotoxic T cell epitopes of common human viruses serving as an internal positive control. The validation determined that first, the overall variation of a positive response of approximately 500 spot-forming units (SFU)/10(6) cells was 21%, while second, the average of 5 SFU/10(6) cells was detected for the seven HIVA-derived pools in HIV-uninfected individuals; third, a positive response to a peptide added to the assay pools was not occluded by the other pool peptides; fourth, the frequencies detected in fresh PBMCs were 2- to 3-fold higher compared with the same samples that had been cryopreserved; and finally, all seven HIV-derived pools induced IFN-gamma responses in PBMCs isolated from HIV-infected individuals. The limits of the validation of assays involving biological responses of living cells are discussed.

Appay V, Zaunders JJ, Papagno L, Sutton J, Jaramillo A, Waters A, Easterbrook P, Grey P et al. 2002. Characterization of CD4(+) CTLs ex vivo. J Immunol, 168 (11), pp. 5954-5958. | Show Abstract

The cytotoxic potential of CD8(+) T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4(+) T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4(+) T cells have been the subject of debate. Here we show that a population of CD4(+) perforin(+) T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4(+) T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4(+) T cells. The existence of CD4(+) cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.

Kaul R, Rowland-Jones SL, Gillespie G, Kimani J, Dong T, Kiama P, Simonsen JN, Bwayo JJ, McMichael AJ, Plummer FA. 2002. Gonococcal cervicitis is associated with reduced systemic CD8+ T cell responses in human immunodeficiency virus type 1-infected and exposed, uninfected sex workers. J Infect Dis, 185 (10), pp. 1525-1529. | Show Abstract | Read more

Neisseria gonorrhoeae cervicitis and human immunodeficiency virus (HIV) type 1 frequently coinfect core transmitter populations, such as female sex workers. Gonococcal cervicitis is associated with increased viral shedding and plasma viremia in HIV-1-infected women and increased HIV-1 susceptibility in uninfected women. We studied the influence of gonococcal cervicitis on CD8(+) interferon (IFN)-gamma responses to HIV-1 and cytomegalovirus (CMV) epitopes in HIV-1-infected and in highly-exposed, persistently seronegative (HEPS) female sex workers. In HIV-1-infected women, gonococcal cervicitis was associated with reduced IFN-gamma responses in bulk CD8(+) lymphocyte populations, and intracellular cytokine staining, combined with class I major histocompatibility complex (MHC)-peptide tetramer studies, demonstrated reduced IFN-gamma production by HIV-1 epitope-specific CD8(+) lymphocytes. In HEPS sex workers, cervicitis was associated with the transient loss of systemic HIV-1-specific CD8(+) responses and with reduced function of CMV-specific CD8(+) lymphocytes. Impaired function of virus-specific CD8(+) lymphocytes may partly explain the deleterious effects of gonococcal cervicitis on HIV-1 immune control and susceptibility.

Hanke T, McMichael AJ, Mwau M, Wee EG, Ceberej I, Patel S, Sutton J, Tomlinson M, Samuel RV. 2002. Development of a DNA-MVA/HIVA vaccine for Kenya. Vaccine, 20 (15), pp. 1995-1998. | Show Abstract | Read more

Without going into the details of the devastation that human immunodeficiency virus (HIV) infection causes especially in the developing world, the best hope for changing the course of this epidemic is development of a safe, effective, accessible prophylactic HIV vaccine. While the inaccessibility of potentially neutralising epitopes on primary HIV isolates has hampered the development of envelope-based vaccines, there is a number of new potent technologies capable of inducing high levels of circulating virus-specific CD8(+) cytotoxic T lymphocytes (CTL). Our original finding that a successive immunisation with DNA and modified vaccinia virus Ankara (MVA) vaccines expressing a common immunogen is a potent way of inducing CD8(+) CTL, which has been since reinforced by us and others, prompted us to test this approach in humans. With the view of proceeding into a high-risk cohort in Kenya for the efficacy trial, we designed the immunogen, termed HIVA, to match the HIV strain responsible locally for over 70% infections. It consists of a consensus clade A gag p24/p17 and a string of clade A-derived CTL epitopes. Pre-clinical studies demonstrated high immunogenicities of both the pTHr.HIVA and MVA.HIVA vaccines. In mice, these induced strong T cells-mediated immune responses which lasted at least 155 days. In rhesus macaques, the prime-boost immunisation elicited T cell responses specific for multiple HIV-derived epitopes. Phase I trials in healthy low-risk volunteers have commenced in Oxford and Nairobi, and the preliminary immunogenicity analysis from the Oxford site indicated that both vaccine components alone induced T cell responses in a majority of volunteers. These results have boosted expectations for the prime-boost vaccinations.

Gillespie GM, Kaul R, Dong T, Yang HB, Rostron T, Bwayo JJ, Kiama P, Peto T, Plummer FA, McMichael AJ, Rowland-Jones SL. 2002. Cross-reactive cytotoxic T lymphocytes against a HIV-1 p24 epitope in slow progressors with B*57. AIDS, 16 (7), pp. 961-972. | Show Abstract | Read more

OBJECTIVES: To determine whether CD8 T lymphocytes from HIV-1-infected patients expressing B*5701 and B*5703 show broad cross-reactivity against different variants of a conserved p24 epitope, which might account for the good prognosis of HIV-1-infected individuals with HLA-B*57. DESIGN: B*5701+ and B*5703+ were recruited from Nairobi, Kenya and from Oxford, UK. All patients had been HIV positive for at least 8 years and could be categorized as slow progressors. METHODS: CD8 cytotoxic T cell clones were generated from B*5701+ and B*5703+ donors and tested for their ability to recognize clade variants of an index p24 epitope in standard cytolytic assays. Cross-reactive responses in freshly isolated peripheral blood mononuclear cells (PBMC) were assessed by interferon-gamma (IFNgamma) production and tetramer binding. RESULTS: Broad cross-clade reactivity for both cytolysis and tetramer binding was observed in CD8 T cell clones from patients harbouring the index epitope sequence. Patterns of cross-reactivity were similar in freshly isolated PBMC but varied between individuals in terms of strength and breath of responses generated. One common variant induced an unusual response with tetramer binding but often failed to induce IFNgamma production, and another was a weak stimulator of both IFNgamma and cytolytic activity. CONCLUSION: B*5701+ and B5703+ donors demonstrate broad functional cross-reactivity to both common and rare variants of a dominant p24 epitope, which could be relevant to the association of B*57 alleles with slow progression to AIDS.

Davenport MP, Fazou C, McMichael AJ, Callan MF. 2002. Clonal selection, clonal senescence, and clonal succession: the evolution of the T cell response to infection with a persistent virus. J Immunol, 168 (7), pp. 3309-3317. | Show Abstract

We have analyzed the CD8(+) T cell response to EBV and find that a larger primary burst size is associated with proportionally greater decay during the development of memory. Consequently, immunodominance and clonal dominance are less marked in memory than primary responses. An intuitive interpretation of this finding is that there is a limit to the number of cell divisions a T cell clone can undergo, and that the progeny of clones that have expanded massively during a primary immune response are more prone to die as a result of senescence. To test this hypothesis, we have derived a mathematical model of the response of different T cell clones of varying avidity for Ag in the primary and persistent phases of viral infection. When cellular survival and replication are linked to T cell avidity for Ag and Ag dose, then high-avidity T cells dominate both the primary and secondary responses. We then incorporated a limit in the number of cell divisions of individual T cell clones to test whether such a constraint could reproduce the observed association between cell division number and alterations in the contribution of clones to the response to persistent infection. Comparison of the model output with the experimental results obtained from primary and persistent EBV infection suggests that there is indeed a role for cellular senescence in shaping the immune response to persistent infection.

Appay V, Papagno L, Spina CA, Hansasuta P, King A, Jones L, Ogg GS, Little S, McMichael AJ, Richman DD, Rowland-Jones SL. 2002. Dynamics of T cell responses in HIV infection. J Immunol, 168 (7), pp. 3660-3666. | Show Abstract

Cytotoxic CD8(+) T cells play a major role in the immune response against viruses. However, the dynamics of CD8(+) T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8(+) T cells, in relation to Ag-specific CD4(+) T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8(+) T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.

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.

Bird LA, Peh CA, Kollnberger S, Sun MY, Elliott T, McMichael AJ, Bowness P. 2002. Identification and characterisation of cell surface HLA-B27 homodimers TISSUE ANTIGENS, 59 pp. 36-36.

Appay V, Hansasuta P, Sutton J, Schrier RD, Wong JK, Furtado M, Havlir DV, Wolinsky SM et al. 2002. Persistent HIV-1-specific cellular responses despite prolonged therapeutic viral suppression. AIDS, 16 (2), pp. 161-170. | Show Abstract | Read more

DESIGN: Antiretroviral therapy (ART) currently represents the best way to avert the lethal consequences of chronic persistent HIV-1 infection. It leads to significant reductions of plasma viremia, often to undetectable levels, but it can also be linked with the reduction and disappearance of detectable HIV-specific CD8 T-cell responses. RESULTS: Here we describe a group of patients in whom ongoing replication of HIV, particularly transcription of Nef mRNA species, was detected despite prolonged and clinically successful antiretroviral treatment. Modest, but significant, numbers of HIV-specific CD8 T cells and CD4 T-cell responses were found in these subjects, with the strongest responses directed towards Nef epitopes. Detailed phenotypic analysis of the HIV-specific CD8 cells demonstrated low perforin levels and persistent expression of CD27, a phenotype associated with incomplete differentiation of cytotoxic T lymphocytes (CTL). CONCLUSION: This immature CTL phenotype has been described previously in association with chronic HIV disease, but its continued persistence is surprising in the setting of prolonged viral suppression on therapy and the presence of HIV-specific CD4 cell activity.

Wee EG, Patel S, McMichael AJ, Hanke T. 2002. A DNA/MVA-based candidate human immunodeficiency virus vaccine for Kenya induces multi-specific T cell responses in rhesus macaques. J Gen Virol, 83 (Pt 1), pp. 75-80. | Show Abstract | Read more

The minimum requirement for candidate human immunodeficiency virus (HIV) vaccines to enter clinical evaluation in humans should be their demonstrable immunogenicity in non-human primates: induction of antibodies neutralizing primary HIV isolates or elicitation of broad T cell-mediated immune responses. Here, we showed in rhesus macaques that the very same vaccines that had entered clinical trials in Oxford and Nairobi, plasmid pTHr.HIVA DNA and recombinant modified vaccinia virus Ankara MVA.HIVA in a prime-boost protocol (Hanke & McMichael, Nature Medicine 6, 951-955, 2000), induced cellular immune responses specific for multiple HIV-derived epitopes. This was demonstrated by using the intracellular cytokine staining and ELISPOT assays detecting interferon-gamma and pools of peptides employed in the clinical studies. These results have both boosted our expectations for the performance of these vaccines in humans and increased our confidence about the choice of these assays as the primary readouts in the on-going human trials.

Gillespie GMA, Appay V, Rowland-Jones SL, McMichael AJ. 2002. The use of tetramers in the quantitative analysis of T-cell responses IMMUNOLOGY OF INFECTION, SECOND EDITION, 32 pp. 125-156.

Ho LP, Slade M, Davies RJO, McMichael AJ. 2001. Characterising the role of dendritic cells (DCs') in the immunopathology of sarcoidosis THORAX, 56 pp. 41-41.

Xu XN, Screaton GR, McMichael AJ. 2001. Virus infections: escape, resistance, and counterattack. Immunity, 15 (6), pp. 867-870. | Show Abstract | Read more

Many viruses establish life-long infections in their natural host with few if any clinical manifestations. The relationship between virus and host is a dynamic process in which the virus has evolved the means to coexist by reducing its visibility, while the host immune system attempts to suppress and eliminate infection without damage to itself. This short review describes a variety of strategies that are employed by viruses to evade host immune responses. These include virus-associated escape from T cell recognition, and resistance to apoptosis and counterattack, with special reference to two papers published in this issue of Immunity (Mueller et al., 2001; Raftery et al., 2001).

Fazou C, Yang H, McMichael AJ, Callan MF. 2001. Epitope specificity of clonally expanded populations of CD8+ T cells found within the joints of patients with inflammatory arthritis. Arthritis Rheum, 44 (9), pp. 2038-2045. | Show Abstract | Read more

OBJECTIVE: To investigate the hypothesis that clonality of synovial T cells from patients with rheumatoid arthritis is at least partly due to the presence of virus-specific T cells expressing a restricted repertoire of T cell receptors (TCRs). METHODS: Using fluorescently labeled HLA class I-peptide tetramers, populations of virus-specific CD8+ T cells were identified in samples of peripheral blood and synovial fluid taken from 4 patients with inflammatory arthritis. The TCR repertoire of the virus-specific T cells in the synovial fluid was analyzed using a panel of TCR beta variable region-specific monoclonal antibodies. Where T cells expressing a particular Vbeta chain dominated the response to a viral epitope, the sequences of these Vbeta chains were derived from sorted populations of antigen-specific T cells by reverse transcription-polymerase chain reaction. RESULTS: CD8+ T cells specific for Epstein-Barr virus, cytomegalovirus, and influenza virus were enriched in synovial fluid compared with peripheral blood. Clonal or oligoclonal populations of CD8+ T cells were found to dominate the responses to these viral epitopes in synovial fluid. CONCLUSION: The results support the hypothesis that restricted T cell receptor usage by large populations of virus-specific T cells provides one explanation for the presence of clonally expanded CD8+ T cells within the joints of patients with inflammatory arthritis. Thus, T cell clonality at a site of inflammation may reflect enrichment for memory T cells specific for foreign antigens, rather than proliferation of autoreactive T cells specific for self antigens.

Kelleher AD, Booth BL, Sewell AK, Oxenius A, Cerundolo V, McMichael AJ, Phillips RE, Price DA. 2001. Effects of retroviral protease inhibitors on proteasome function and processing of HIV-derived MHC class I-restricted cytotoxic T lymphocyte epitopes. AIDS Res Hum Retroviruses, 17 (11), pp. 1063-1066. | Read more

van Baarle D, Hovenkamp E, Callan MF, Wolthers KC, Kostense S, Tan LC, Niesters HG, Osterhaus AD, McMichael AJ, van Oers MH, Miedema F. 2001. Dysfunctional Epstein-Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDS-related non-Hodgkin lymphoma. Blood, 98 (1), pp. 146-155. | Show Abstract | Read more

Acquired immunodeficiency syndrome-related non-Hodgkin lymphomas (AIDS-NHL) are thought to arise because of loss of Epstein-Barr Virus (EBV)-specific cellular immunity. Here, an investigation was done to determine whether cellular immunity to EBV is lost because of physical loss or dysfunction of EBV-specific cytotoxic T cells. Data on EBV-specific cellular immunity were correlated with EBV load. For comparison, individuals who progressed to AIDS with opportunistic infections (AIDS-OI) and long-term asymptomatics (LTAs) were studied. The number of virus-specific T cells was detected using tetrameric HLA-EBV-peptide complexes; function of these EBV-specific T cells was determined using the interferon-gamma (IFN-gamma) Elispot assay. It was observed that EBV-specific CD8(+) T cells were present in normal numbers in human immunodeficiency virus (HIV)-infected individuals. However, their functional capacity was decreased compared with HIV(-) individuals. In AIDS-NHL patients, EBV-specific T cells were not physically lost in the course of HIV-1 infection but showed progressive loss of their capability to produce IFN-gamma in response to EBV peptides. This loss of function correlated with lower CD4(+) T-cell numbers and was accompanied by increasing EBV load. In HIV-1-infected LTA individuals, in whom CD4(+) T-cell numbers were maintained, and progressors to AIDS-OI, IFN-gamma-producing EBV-specific T cells were stable and EBV load remained stable or decreased in the course of HIV infection, suggestive of immune control. Our data indicate that functional loss of EBV-specific CD8(+) T cells with a concomitant increase in EBV load may play a role in the pathogenesis of AIDS-NHL.

Wodarz D, Hall SE, Usuku K, Osame M, Ogg GS, McMichael AJ, Nowak MA, Bangham CR. 2001. Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1. Proc Biol Sci, 268 (1473), pp. 1215-1221. | Show Abstract | Read more

The correlation between virus load and specific cytotoxic T-lymphocyte (CTL) frequency during the chronic phase in human immunodeficiency virus type 1 (HIV-1) infection has been found to be negative in cross-sectional studies. We report here that, in infection with the related retrovirus human T-cell leukaemia virus type 1 (HTLV-1), the correlation is positive in asymptomatic carriers and zero in patients with the associated inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We demonstrate that the direction of the correlation may depend on the efficacy of the CTL response using mathematical models. We conclude that the CTL response is effective in asymptomatic carriers of HTLV-1, but ineffective in patients with HAM/TSP. Virus-mediated impairment of specific CTL production in HIV-1 infection can account for the negative correlation observed.

Simmons A, Aluvihare V, McMichael A. 2001. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity, 14 (6), pp. 763-777. | Show Abstract | Read more

Gene expression profiling was used to explore the role of Nef in HIV. Nef induces a transcriptional program in T cells that is 97% identical to that of anti-CD3 T cell activation. This program is inhibited in the presence of cyclosporin. A requirement for TCR zeta and ZAP-70 is demonstrated for formation of the complete profile. Among eight factors particular to the anti-CD3 activation profile are IL16 and YY1, negative regulators of HIV transcription. In contrast, Nef exclusively upregulates factors positively regulating HIV, including Tat-SF1, U1 SNRNP, and IRF-2. New genes associated with Nef include CDK9, the induction of which enhances Tat function. Thus, Nef acts as a master switch early in the viral life cycle, forcing an environment conducive to dynamic viral production.

Kaul R, Dong T, Plummer FA, Kimani J, Rostron T, Kiama P, Njagi E, Irungu E et al. 2001. CD8(+) lymphocytes respond to different HIV epitopes in seronegative and infected subjects. J Clin Invest, 107 (10), pp. 1303-1310. | Show Abstract | Read more

HIV-1-specific cytotoxic T-lymphocyte (CTL) responses have been detected at a low frequency in many HIV-1-exposed, persistently seronegative (HEPS) subjects. However, it is unclear how CTLs could protect against HIV acquisition in HEPS subjects, when high levels of circulating CTL fail to prevent disease progression in most seropositive subjects. To address this issue we studied CD8(+) lymphocyte responses to a panel of HIV-1 CTL epitopes in 91 HEPS and 87 HIV-1-infected Nairobi sex workers. HIV-specific responses in seropositive women focused strongly on epitopes rarely or never recognized in HEPS subjects, who targeted epitopes that were subdominant or unrecognized in infected women. These differences in epitope specificity were restricted by only those HLA class I alleles that are associated with a reduced risk of HIV-1 infection in this cohort. Late seroconversion in HEPS donors was associated with a switch in epitope specificity and/or immunodominance to those epitopes preferentially recognized by HIV-1-infected women. The likelihood of detecting HIV-1-specific responses in HEPS women increased with the duration of viral exposure, suggesting that HIV-1-specific CD8(+) responses are acquired over time. The association between differential recognition of distinct CTL epitopes and protection from HIV-1 infection may have significant implications for vaccine design.

Xu XN, Purbhoo MA, Chen N, Mongkolsapaya J, Cox JH, Meier UC, Tafuro S, Dunbar PR et al. 2001. A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex. Immunity, 14 (5), pp. 591-602. | Show Abstract | Read more

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

McMichael AJ, Rowland-Jones SL. 2001. Cellular immune responses to HIV. Nature, 410 (6831), pp. 980-987. | Show Abstract | Read more

The cellular immune response to the human immunodeficiency virus, mediated by T lymphocytes, seems strong but fails to control the infection completely. In most virus infections, T cells either eliminate the virus or suppress it indefinitely as a harmless, persisting infection. But the human immunodeficiency virus undermines this control by infecting key immune cells, thereby impairing the response of both the infected CD4+ T cells and the uninfected CD8+ T cells. The failure of the latter to function efficiently facilitates the escape of virus from immune control and the collapse of the whole immune system.

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Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, Appay V, Rizzardi GP et al. 2001. Skewed maturation of memory HIV-specific CD8 T lymphocytes NATURE, 410 (6824), pp. 106-111. | Read more

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Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, Conlon C, Workman C, Shaunak S et al. 2001. Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses JOURNAL OF EXPERIMENTAL MEDICINE, 193 (3), pp. 375-385. | Show Abstract | Read more

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263-272, KR WIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

Kelleher AD, Long C, Holmes EC, Allen RL, Wilson J, Conlon C, Workman C, Shaunak S et al. 2001. Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses. J Exp Med, 193 (3), pp. 375-386. | Show Abstract | Read more

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263-272, KRWIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

Kaul R, Rowland-Jones SL, Kimani J, Dong T, Yang HB, Kiama P, Rostron T, Njagi E et al. 2001. Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses. J Clin Invest, 107 (3), pp. 341-349. | Show Abstract | Read more

Resistance to HIV infection in a small group of Kenyan sex workers is associated with CD8+-lymphocyte responses to HIV cytotoxic T-lymphocyte (CTL) epitopes. Eleven prostitutes meeting criteria for HIV resistance seroconverted between 1996 and 1999. The occurrence and specificity of preexisting HIV-1 epitope-specific responses were examined using the IFN-gamma enzyme-linked immunospot assay, and any epitopes recognized were cloned and sequenced from the infecting viral isolate. Immunologic and behavioral variables were compared between late seroconverters and persistently uninfected sex worker controls. HIV-1 CTL epitope responses were present in four of six cases, 5-18 months before seroconversion, and their presence was confirmed by bulk CTL culture. A possible viral escape mutation was found in one of six epitopes. The key epidemiologic correlate of late seroconversion was a reduction in sex work over the preceding year. In persistently uninfected controls, a break from sex work was associated with a loss of HIV-specific CD8+ responses. Late seroconversion may occur in HIV-1-resistant sex workers despite preceding HIV-specific CD8+ responses. Seroconversion generally occurs in the absence of detectable CTL escape mutations and may relate to the waning of HIV-specific CD8+ responses due to reduced antigenic exposure.

Tafuro S, Meier UC, Dunbar PR, Jones EY, Layton GT, Hunter MG, Bell JI, McMichael AJ. 2001. Reconstitution of antigen presentation in HLA class I-negative cancer cells with peptide-beta2m fusion molecules. Eur J Immunol, 31 (2), pp. 440-449. | Show Abstract | Read more

Engineered MHC-peptide targets capable of inducing recognition by CTL may prove useful in designing vaccines for infectious disease and cancer. We tested whether peptides directly linked to beta2-microglobulin (beta2m) could complex with human HLA class I heavy chain, and could be recognized by human CTL, both as soluble reagents and as cell surface constituents. An HLA-A2-restricted peptide epitope was physically linked to the N terminus of human beta2m. This fusion protein refolded efficiently in vitro with HLA-A2 heavy chain, and when multimerized, the resultant complexes ("fusamers") bound specifically to appropriate CTL clones. These fused peptide/MHC complexes were as efficient as standard tetrameric peptide/MHC complexes in recognizing antigen-specific CTL. When the fusion protein was delivered to target cells using a retroviral vector, these cells were recognized and killed by appropriate CTL clones. Efficient sensitization to CTL lysis was achieved in TAP-negative and beta2m-negative cell lines, as well as in unmutated B cell lines, proving that such constructs may be effective in inducing CTL even when the MHC class I pathway has been disrupted. Specific peptides covalently linked to beta2m and delivered via retroviral vectors may be useful reagents for in vivo priming of CTL against epitopes of clinical relevance.

Lepin EJ, Bastin JM, Allan DS, Roncador G, Braud VM, Mason DY, van der Merwe PA, McMichael AJ, Bell JI, Powis SH, O'Callaghan CA. 2000. Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. Eur J Immunol, 30 (12), pp. 3552-3561. | Show Abstract | Read more

HLA-F is a human non-classical MHC molecule. Recombinant HLA-F heavy chain was refolded with 2-microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high-affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA-F. HLA-F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines and from HUT-78, a T cell line. HLA-F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA-F tetramers stain peripheral blood monocytes and B cells. HLA-F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA-F, suggest that HLA-F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.

Callan MF, Fazou C, Yang H, Rostron T, Poon K, Hatton C, McMichael AJ. 2000. CD8(+) T-cell selection, function, and death in the primary immune response in vivo. J Clin Invest, 106 (10), pp. 1251-1261. | Show Abstract | Read more

The primary immune response to Epstein Barr virus (EBV) is characterized by striking proliferation of EBV-specific CD8(+) T cells. In this study we have investigated the clonal composition and functional properties of the cells mediating this primary response and have analyzed the mechanisms that control the downregulation of the primary response and the selection of memory cells. We show that massively expanded T-cell clones often dominate the primary antigen-specific T-cell response. Despite the enormous extent of expansion, the virus-specific T cells express high levels of intracellular perforin and are potently cytotoxic. They are, however, functionally heterogeneous in their ability to secrete proinflammatory cytokines, with subpopulations of the antigen-specific T cells being hyporesponsive. The primary response is closely regulated, and the majority of cells are programmed to die via a cytokine-rescuable pathway, leaving only small populations of memory T cells surviving. Comparison of the clonal composition of primary and memory responses in vivo shows that the clones that dominate the primary response are relatively heavily culled during the downregulation of the primary response and the establishment of T-cell memory.

Tomiyama H, Oka S, Ogg GS, Ida S, McMichael AJ, Takiguchi M. 2000. Expansion of HIV-1-specific CD28- CD45RA- CD8+ T cells in chronically HIV-1-infected individuals. AIDS, 14 (13), pp. 2049-2051. | Read more

Hanke T, McMichael AJ. 2000. Design and construction of an experimental HIV-1 vaccine for a year-2000 clinical trial in Kenya. Nat Med, 6 (9), pp. 951-955. | Read more

McMichael AJ, Callan M, Appay V, Hanke T, Ogg G, Rowland-Jones S. 2000. The dynamics of the cellular immune response to HIV infection: implications for vaccination. Philos Trans R Soc Lond B Biol Sci, 355 (1400), pp. 1007-1011. | Show Abstract | Read more

Recent advances in measuring T-cell responses to viruses have led to new insights into how these T cells respond. In the acute infection there are massive CD8+ T-cell responses to both Epstein-Barr virus (EBV) and to human immunodeficiency virus (HIV). Many of these T cells are effector cells and only a minority appear to be capable of maintaining immunological memory. In persistent virus infections, high levels of antigen-specific effector cells persist. If virus does not persist, the effectors fade in number but memory is maintained and is primed to react rapidly to a new challenge. A vaccine that stimulates only T-cell responses may protect when these memory cells respond rapidly enough to generate high numbers of effectors before the infecting virus becomes established.

Appay V, Nixon DF, Donahoe SM, Gillespie GM, Dong T, King A, Ogg GS, Spiegel HM et al. 2000. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J Exp Med, 192 (1), pp. 63-75. | Show Abstract | Read more

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

King A, Allan DS, Bowen M, Powis SJ, Joseph S, Verma S, Hiby SE, McMichael AJ, Loke YW, Braud VM. 2000. HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Eur J Immunol, 30 (6), pp. 1623-1631. | Show Abstract | Read more

Non-classical MHC class I molecule HLA-E is the ligand for CD94/NKG2 NK cell receptors. Surface expression of HLA-E requires binding of specific HLA class I leader sequences. The uterine mucosa in early pregnancy (decidua) is infiltrated by large numbers of NK cells, which are closely associated with placental trophoblast cells. In this study we demonstrate that trophoblast cells express HLA-E on their cell surface in addition to the previously reported expression of HLA-G and HLA-C. Furthermore, we show that the vast majority of decidual NK cells bind to HLA-E tetrameric complexes and this binding is inhibited by mAb to CD94. Thus, recognition of fetal HLA-E by decidual NK cells may play a key role in regulation of placentation. The functional consequences of decidual NK cell interaction were investigated in cytotoxicity assays using polyclonal decidual NK cells. The overall effect of CD94/NKG2 interaction with HLA-E is inhibition of cytotoxicity by decidual NK cells. However, since decidual NK cells are unable to kill trophoblast even in the presence of mAb to MHC class I molecules and NK cell receptors, HLA-E interaction with CD94/NKG2 receptors may regulate other functions besides cytolysis during implantation.

Allen TM, Vogel TU, Fuller DH, Mothé BR, Steffen S, Boyson JE, Shipley T, Fuller J et al. 2000. Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen. J Immunol, 164 (9), pp. 4968-4978. | Show Abstract

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

Allan DS, McMichael AJ, Braud VM. 2000. The ILT family of leukocyte receptors. Immunobiology, 202 (1), pp. 34-41. | Show Abstract | Read more

In this short review we examine the recently identified immunoglobulin-like transcript (ILT) family of receptors (also known as LIR and MIR). ILT are expressed by many leukocyte subsets, especially monocytic cells. Expression levels of certain ILT molecules allow definition of blood monocyte and dendritic cell (DC) subsets. Two receptors, ILT2 and ILT4, recognise a broad range of MHC class I molecules and transduce an inhibitory signal. Such recognition may give many cell lineages the potential to recognise and respond to MHC class I down-regulation.

McMichael AJ, Ogg G, Wilson J, Callan M, Hambleton S, Appay V, Kelleher T, Rowland-Jones S. 2000. Memory CD8+ T cells in HIV infection. Philos Trans R Soc Lond B Biol Sci, 355 (1395), pp. 363-367. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent HIV infection in humans. The kinetics and general features of the CTL response are similar to those found during other persisting virus infections in humans. During chronic infection there are commonly between 0.1 and 1.0% of all CD8+ T cells in the blood that are specific for immunodominant virus epitopes, as measured by HLA class I peptide tetramers. These figures are greatly in excess of the numbers found by limiting dilution assays; the discrepancy may arise because in the latter assay, CTLs have to divide many times to be detected and many of the HIV-specific CD8+ T cells circulating in infected persons may be incapable of further division. Many tetramer-positive T cells make interferon-gamma, beta-chemokines and perforin, so are probably functional. It is not known how fast these T cells turn over, but in the absence of antigen they decay in number. Impairment of CTL replacement, because CD4+ T helper cells are depleted by HIV infection, may play a major role in the development of AIDS.

Ogg GS, Dunbar PR, Cerundolo V, McMichael AJ, Lemoine NR, Savage P. 2000. Sensitization of tumour cells to lysis by virus-specific CTL using antibody-targeted MHC class I/peptide complexes. Br J Cancer, 82 (5), pp. 1058-1062. | Show Abstract | Read more

A number of cell surface molecules with specificity to tumour cells have been identified and monoclonal antibodies (mAb) to some of these antigens have been used for targeting tumour cells in vivo. We have sought to link the powerful effector mechanisms of cytotoxic T-cells with the specificity of mAb, by targeting recombinant HLA class I molecules to tumour cells using an antibody delivery system. Soluble recombinant MHC class I/peptide complexes including HLA-A2.1 refolded around an immunodominant peptide from the HIV gag protein (HLA-A2/gag) were synthesized, and the stability of these complexes at 37 degrees C was confirmed by enzyme-linked immunosorbent assay using a conformation-specific antibody. MHC class I-negative lymphoma cells (Daudi) were labelled with a biotinylated mAb specific for a cell surface protein (anti-CD20) then linked to soluble biotinylated HLA-A2/gag complexes using an avidin bridge. Flow cytometry revealed strong labelling of lymphoma cells with HLA-A2/gag complexes (80-fold increase in mean channel fluorescence). CTL specific for HLA-A2/gag efficiently lysed complex-targeted cells, while control CTL (specific for an HLA-A2.1-restricted epitope of melan-A) did not. Similarly, SK-mel-29 melanoma cells were also efficiently lysed by HLA-A2/gag-specific CTL when HLA-A2/gag complexes were linked to their surface via the HMW-MAA specific anti-melanoma antibody 225.28s. With further consideration to the in vivo stability of the MHC class I/peptide complexes, this system could prove a new strategy for the immunological therapy of cancer.

Wilson JD, Ogg GS, Allen RL, Davis C, Shaunak S, Downie J, Dyer W, Workman C, Sullivan S, McMichael AJ, Rowland-Jones SL. 2000. Direct visualization of HIV-1-specific cytotoxic T lymphocytes during primary infection. AIDS, 14 (3), pp. 225-233. | Show Abstract | Read more

OBJECTIVE: HIV-specific cytotoxic T lymphocytes (CTL) are believed to play an important role in containing viral replication throughout HIV-1 infection. Previous studies have attempted to quantify the HIV-1-specific CTL precursor frequency during primary HIV infection by using limiting dilution analysis, which almost certainly underestimates the true CTL frequency. Here we use a relatively new technique to quantify HIV-specific CD8 T cells in primary HIV infection. METHODS: We have used soluble tetrameric complexes of HLA class I molecules complexed with HIV epitope peptides to study the dynamics and frequency of HIV-specific CD8 T cells in relation to plasma viral load in early HIV infection, in three patients with a highly focused HIV-specific CTL response. RESULTS: We show that the frequencies of HIV-1-specific CD8 T cells in acute infection are significantly higher than previously documented and can be demonstrated well before full seroconversion. These studies also confirm the immunodominance of the B27-restricted response in HIV infection and demonstrate a close temporal relationship between the numbers of circulating HIV-specific CD8 T cells and viral load. CONCLUSIONS: These findings strongly suggest that HIV-1-specific CD8 T cells are responding directly to the level of viral replication in early HIV infection and are a major factor in its control. In addition, the data indicate that immunodominance for CD8 T-cell responses is established in the acute phase of HIV infection.

Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, Gadola S, Cerundolo V, Borysiewicz LK, McMichael AJ, Wilkinson GW. 2000. Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science, 287 (5455), pp. 1031. | Show Abstract | Read more

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

Kaul R, Plummer FA, Kimani J, Dong T, Kiama P, Rostron T, Njagi E, MacDonald KS, Bwayo JJ, McMichael AJ, Rowland-Jones SL. 2000. HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi. J Immunol, 164 (3), pp. 1602-1611. | Show Abstract

Understanding how individuals with a high degree of HIV exposure avoid persistent infection is paramount to HIV vaccine design. Evidence suggests that mucosal immunity, particularly virus-specific CTL, could be critically important in protection against sexually acquired HIV infection. Therefore, we have looked for the presence of HIV-specific CD8+ T cells in cervical mononuclear cells from a subgroup of highly HIV-exposed but persistently seronegative female sex workers in Nairobi. An enzyme-linked immunospot assay was used to measure IFN-gamma release in response to known class I HLA-restricted CTL epitope peptides using effector cells from the blood and cervix of HIV-1-resistant and -infected sex workers and from lower-risk uninfected controls. Eleven of 16 resistant sex workers had HIV-specific CD8+ T cells in the cervix, and a similar number had detectable responses in blood. Where both blood and cervical responses were detected in the same individual, the specificity of the responses was similar. Neither cervical nor blood responses were detected in lower-risk control donors. HIV-specific CD8+ T cell frequencies in the cervix of HIV-resistant sex workers were slightly higher than in blood, while in HIV-infected donor cervical response frequencies were markedly lower than blood, so that there was relative enrichment of cervical responses in HIV-resistant compared with HIV-infected donors. HIV-specific CD8+ T cell responses in the absence of detectable HIV infection in the genital mucosa of HIV-1-resistant sex workers may be playing an important part in protective immunity against heterosexual HIV-1 transmission.

Spiegel HM, Ogg GS, DeFalcon E, Sheehy ME, Monard S, Haslett PA, Gillespie G, Donahoe SM et al. 2000. Human immunodeficiency virus type 1- and cytomegalovirus-specific cytotoxic T lymphocytes can persist at high frequency for prolonged periods in the absence of circulating peripheral CD4(+) T cells. J Virol, 74 (2), pp. 1018-1022. | Show Abstract | Read more

CD4(+) T cells are thought to be critical in the maintenance of virus-specific CD8(+) cytotoxic T-cell (CTL) responses. In human immunodeficiency virus type 1 (HIV-1) infection, a selective decline in HIV-1-specific CTL as the CD4(+) T-cell count decreases has been reported. Using HLA-peptide tetrameric complexes, we show the presence at high frequency of HIV-1- and cytomegalovirus-specific CD8(+) T cells when the peripheral CD4(+) T-cell count was low or zero in three HIV-1-infected patients. No direct virus-specific CD8(+)-mediated effector activity was seen in these subjects, suggesting antigen unresponsiveness, although tetramer-sorted cells could be expanded in vitro in the presence of interleukin-2 into responsive effector cells. Thus, virus-specific CD8(+) T cells can be maintained in the peripheral circulation at high frequency in the absence of circulating peripheral CD4(+) T cells, but these cells may lack direct effector activity. Strategies designed to overcome this antigen unresponsiveness may be of value in therapies for the treatment of AIDS.

Tan LC, Mowat AG, Fazou C, Rostron T, Roskell H, Dunbar PR, Tournay C, Romagné F et al. 2000. Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes. Arthritis Res, 2 (2), pp. 154-164. | Show Abstract | Read more

INTRODUCTION: Epstein-Barr virus (EBV) is transmitted orally, replicates in the oropharynx and establishes life-long latency in human B lymphocytes. T-cell responses to latent and lytic/replicative cycle proteins are readily detectable in peripheral blood from healthy EBV-seropositive individuals. EBV has also been detected within synovial tissue, and T-cell responses to EBV lytic proteins have been reported in synovial fluid from a patient with rheumatoid arthritis (RA). This raises the question regarding whether T cells specific for certain viruses might be present at high frequencies within synovial fluid and whether such T cells might be activated or able to secrete cytokines. If so, they might play a 'bystander' role in the pathogenesis of inflammatory joint disease. OBJECTIVES: To quantify and characterize T cells that are specific for epitopes from EBV, cytomegalovirus (CMV) and influenza in peripheral blood and synovial fluid from patients with arthritis. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from patients with inflammatory arthritis (including those with RA, osteoarthritis, psoriatic arthritis and reactive arthritis). Samples from human leucocyte antigen (HLA)-A2-positive donors were stained with fluorescent-labelled tetramers of HLA-A2 complexed with the GLCTLVAML peptide epitope from the EBV lytic cycle protein BMLF1, the GILGFVFTL peptide epitope from the influenza A matrix protein, or the NLVPMVATV epitope from the CMV pp65 protein. Samples from HLA-B8-positive donors were stained with fluorescent-labelled tetramers of HLA-B8 complexed with the RAKFKQLL peptide epitope from the EBV lytic protein BZLF1 or the FLRGRAYGL peptide epitope from the EBV latent protein EBNA3A. All samples were costained with an antibody specific for CD8. CD4+ T cells were not analyzed. Selected samples were costained with antibodies specific for cell-surface glycoproteins, in order to determine the phenotype of the T cells within the joint and the periphery. Functional assays to detect release of IFN- or tumour necrosis factor (TNF)- were also performed on some samples. RESULTS: The first group of 15 patients included 10 patients with RA, one patient with reactive arthritis, one patient with psoriatic arthritis and three patients with osteoarthritis. Of these, 11 were HLA-A2 positive and five were HLA-B8 positive. We used HLA-peptide tetrameric complexes to analyze the frequency of EBV-specific T cells in PBMCs and SFMCs (Figs 1 and 2). Clear enrichment of CD8+ T cells specific for epitopes from the EBV lytic cycle proteins was seen within synovial fluid from almost all donors studied, including patients with psoriatic arthritis and osteoarthritis and those with RA. In donor RhA6, 9.5% of CD8+ SFMCs were specific for the HLA-A2 restricted GLCTLVAML epitope, compared with 0.5% of CD8+ PBMCs. Likewise in a donor with osteoarthritis (NR4), 15.5% of CD8+ SFMCs were specific for the HLA-B8-restricted RAKFKQLL epitope, compared with 0.4% of CD8+ PBMCs. In contrast, we did not find enrichment of T cells specific for the HLA-B8-restricted FLRGRAYGL epitope (from the latent protein EBNA3A) within SFMCs compared with PBMCs in any donors. In selected individuals we performed ELISpot assays to detect IFN- secreted by SFMCs and PBMCs after a short incubation in vitro with peptide epitopes from EBV lytic proteins. These assays confirmed enrichment of T cells specific for epitopes from EBV lytic proteins within synovial fluid and showed that subpopulations of these cells were able to secrete proinflammatory cytokines after short-term stimulation. We used a HLA-A2/GILGFVFTL tetramer to stain PBMCs and SFMCs from six HLA-A2-positive patients. The proportion of T cells specific for this influenza epitope was low (<0.2%) in all donors studied, and we did not find any enrichment within SFMCs. We had access to SFMCs only from a second group of four HLA-A2-positive patients with RA. A tetramer of HLA-A2 complexed to the NLVPMVATV epitope from the CMV pp65 protein reacted with subpopulations of CD8+ SFMCs in all four donors, with frequencies of 0.2, 0.5, 2.3 and 13.9%. SFMCs from all four donors secreted TNF after short-term incubation with COS cells transfected with HLA-A2 and pp65 complementary DNA. We analyzed the phenotype of virus-specific cells within PBMCs and SFMCs in three donors. The SFMC virus-specific T cells were more highly activated than those in PBMCs, as evidenced by expression of high levels of CD69 and HLA-DR. A greater proportion of SFMCs were CD38+, CD62L low, CD45RO bright, CD45RA dim, CD57+ and CD28- when compared with PBMCs. DISCUSSION: This work shows that T cells specific for certain epitopes from viral proteins are present at very high frequencies (up to 15.5% of CD8+ T cells) within SFMCs taken from patients with inflammatory joint disease. This enrichment does not reflect a generalized enrichment for the 'memory pool' of T cells; we did not find enrichment of T cells specific for the GILGFVFTL epitope from influenza A or for the FLRGRAYGL epitope from the EBV latent protein EBNA3A, whereas we found clear enrichment of T cells specific for the GLCTLVAML epitope from the EBV lytic protein BMLF1 and for the RAKFKQLL epitope from the EBV lytic protein BZLF1. The enrichment might reflect preferential recruitment of subpopulations of virus-specific T cells, perhaps based on expression of selectins, chemokine receptors or integrins. Alternatively, T cells specific for certain viral epitopes may be stimulated to proliferate within the joint, by viral antigens themselves or by cross-reactive self-antigens. Finally, it is theoretically possible that subpopulations of T cells within the joint are preferentially protected from apoptotic cell death. Whatever the explanation, the virus-specific T cells are present at high frequency, are activated and are able to secrete proinflammatory cytokines. They could potentially interact with synoviocytes and contribute to the maintenance of inflammation within joints in many different forms of inflammatory arthritis.

Maini MK, Boni C, Ogg GS, King AS, Reignat S, Lee CK, Larrubia JR, Webster GJ et al. 1999. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology, 117 (6), pp. 1386-1396. | Show Abstract | Read more

BACKGROUND & AIMS: Cytotoxic T cells have been suggested to be responsible for lysis of hepatitis B virus (HBV)-infected hepatocytes and control of virus infection. The frequency, kinetics, phenotype, and capacity for clonal expansion of circulating HBV-specific CD8 cells were analyzed directly in patients with acute HBV infection to clarify their pathogenetic role. METHODS: Three HLA-A2 peptide tetramers able to visualize HBV core, envelope, and polymerase epitope-specific cytotoxic T lymphocytes were synthesized and used for flow cytometric analysis of antigen-specific populations. RESULTS: Tetramer-positive cells specific for the core 18-27 epitope were found at a higher frequency than those specific for polymerase 575-583 and envelope 335-343 epitopes in most patients with acute HBV. The number of HBV-specific CD8 cells was highest during the clinically acute stage of infection and decreased after recovery. These cells expressed an activated phenotype and had an impaired capacity to expand in vitro and to display cytolytic activity in response to peptide stimulation. Recovery of these functions was observed when the frequency of specific CD8 cells decreased, coincident with a progressive decrease in their expression of activation markers. CONCLUSIONS: This study provides the first ex vivo evidence that the highest frequency of circulating HBV-specific CD8 cells coincides with the clinically acute phase of hepatitis B. These cells exhibit an activated phenotype with limited further proliferative capacity that is restored during recovery.

McMichael AJ, Kelleher A. 1999. The arrival of HLA class II tetramers. J Clin Invest, 104 (12), pp. 1669-1670. | Read more

Thomson KJ, Manis J, Jones M, Alsheikhly R, Leung R, Davidson L, McMichael AJ, Alt FW, Rahemtulla A. 1999. An in vivo model of reverse signalling through CD40L. BLOOD, 94 (10), pp. 59B-59B.

Ogg GS, Kostense S, Klein MR, Jurriaans S, Hamann D, McMichael AJ, Miedema F. 1999. Longitudinal phenotypic analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes: correlation with disease progression. J Virol, 73 (11), pp. 9153-9160. | Show Abstract

Few studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8(+) T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17(Gag/Pol) tetramer-binding cells and slower disease progression (P < 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27(+) CD45RO(+) cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27(+) CD45RO(+) subset.

Allen RL, Bowness P, McMichael A. 1999. The role of HLA-B27 in spondyloarthritis. Immunogenetics, 50 (3-4), pp. 220-227. | Show Abstract | Read more

The human major histocompatibility complex (MHC) class I allele HLA-B27 bears a striking association with the spondylolarthritic group of inflammatory arthritides, yet despite extensive studies its role in the disease process remains obscure. As an MHC class I protein, the primary function of HLA-B27 is to complex with beta(2)-microglobulin forming a structure that presents short antigenic peptides for recognition by cytotoxic T lymphocytes (CTL). It has been proposed that the role of HLA-B27 in spondyloarthropathy involves this process of antigen presentation, and of the numerous theories proposed to explain the association, the most popular have involved the binding and presentation of "arthritogenic" peptides. Transgenic rodent studies directly implicate HLA-B27 heavy chains in disease pathogenesis, but suggest that the mechanism may be distinct from their primary function. The recent demonstration that HLA-B27 heavy chains can form stable homodimers may thus be of relevance. This review summarizes the evidence supporting current theories of disease association and proposes an alternative model of disease based on recent findings.

Ogg GS, King AS, Dunbar PR, McMichael AJ. 1999. Isolation of HIV-1-specific cytotoxic T lymphocytes using human leukocyte antigen-coated beads. AIDS, 13 (14), pp. 1991-1993. | Read more

Hanke T, Samuel RV, Blanchard TJ, Neumann VC, Allen TM, Boyson JE, Sharpe SA, Cook N et al. 1999. Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen. J Virol, 73 (9), pp. 7524-7532. | Show Abstract

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.

McMichael AJ, Hanke T. 1999. Is an HIV vaccine possible? Nat Med, 5 (6), pp. 612-614. | Read more

Larsson M, Jin X, Ramratnam B, Ogg GS, Engelmayer J, Demoitie MA, McMichael AJ, Cox WI, Steinman RM, Nixon D, Bhardwaj N. 1999. A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS, 13 (7), pp. 767-777. | Show Abstract | Read more

OBJECTIVES: HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. RESULTS: In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. CONCLUSIONS: Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.

Xu XN, Laffert B, Screaton GR, Kraft M, Wolf D, Kolanus W, Mongkolsapay J, McMichael AJ, Baur AS. 1999. Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain. J Exp Med, 189 (9), pp. 1489-1496. | Show Abstract | Read more

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

Allen RL, O'Callaghan CA, McMichael AJ, Bowness P. 1999. Cutting edge: HLA-B27 can form a novel beta 2-microglobulin-free heavy chain homodimer structure. J Immunol, 162 (9), pp. 5045-5048. | Show Abstract

HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular alpha 1 domain. Despite the absence of beta 2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in beta 2-microglobulin-deficient mice.

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Allan DSJ, Colonna M, Lanier LL, Churakova TD, Abrams JS, Ellis SA, McMichael AJ, Braud VM. 1999. Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells JOURNAL OF EXPERIMENTAL MEDICINE, 189 (7), pp. 1149-1155. | Read more

Allan DS, Colonna M, Lanier LL, Churakova TD, Abrams JS, Ellis SA, McMichael AJ, Braud VM. 1999. Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G bind to peripheral blood myelomonocytic cells. J Exp Med, 189 (7), pp. 1149-1156. | Show Abstract | Read more

The nonclassical MHC class I molecule human histocompatibility leukocyte antigen (HLA)-G is selectively expressed on fetal trophoblast tissue at the maternal-fetal interface in pregnancy. It has long been suggested that HLA-G may inhibit maternal natural killer (NK) cells through interaction with particular NK cell receptors (KIRs). To investigate interactions of HLA-G, we constructed phycoerythrin-labeled tetrameric complexes of HLA-G refolded with a self-peptide. These HLA-G tetramers failed to bind to NK cells and cells transfected with CD94/NKG2 and killer immunoglobulin-like NK receptors. In contrast, HLA-G tetramers did bind to peripheral blood monocytes, staining a CD16(+)CD14(mid) subset with greater intensity. On transfectants, HLA-G tetramers bound to inhibitory immunoglobulin-like transcript (ILT)2 and ILT4 receptors. However, staining in the presence of antibodies reactive with ILT receptors revealed that the interaction of HLA-G tetramers with blood monocytes was largely due to binding to ILT4. These results suggest that the primary role of HLA-G may be the modulation of myelomonocytic cell behavior in pregnancy.

Maini MK, Webster GJM, Boni C, Ogg GS, Lee CK, Brown D, Reignat S, Dusheiko GM et al. 1999. Kinetics of the HBV-specific CTL response GUT, 44 pp. A52-A52.

Tan R, Xu X, Ogg GS, Hansasuta P, Dong T, Rostron T, Luzzi G, Conlon CP, Screaton GR, McMichael AJ, Rowland-Jones S. 1999. Rapid death of adoptively transferred T cells in acquired immunodeficiency syndrome. Blood, 93 (5), pp. 1506-1510. | Show Abstract

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) probably play the major role in controlling HIV replication. However, the value of adoptive transfer of HIV-specific CTL expanded in vitro to HIV+ patients has been limited: this contrasts with the success of CTL therapy in treating or preventing Epstein-Barr virus and cytomegalovirus disease after bone marrow transplantation (BMT). We investigated the fate of expanded HIV-specific CTL clones in vivo following adoptive transfer to a patient with acquired immunodeficiency syndrome (AIDS). Two autologous CTL clones specific for HIV Gag and Pol were expanded to large numbers (>10(9)) in vitro and infused into an HIV-infected patient whose viral load was rising despite antiretroviral therapy. The fate of one clone was monitored by staining peripheral blood mononuclear cells (PBMCs) with T-cell receptor-specific tetrameric major histocompatibility complex (MHC)-peptide complexes. Although the CTL transfer was well tolerated, there were no significant changes in CD4 and CD8 lymphocyte counts and virus load. By tracking an infused clone using soluble MHC-peptide complexes, we show that cells bearing the Gag-specific T-cell receptors were rapidly eliminated within hours of infusion through apoptosis. Thus, the failure of adoptively transferred HIV-specific CTL to reduce virus load in AIDS may be due to rapid apoptosis of the infused cells, triggered by a number of potential mechanisms. Further trials of adoptive transfer of CTL should take into account the susceptibility of infused cells to in vivo apoptosis.

Tan LC, Gudgeon N, Annels NE, Hansasuta P, O'Callaghan CA, Rowland-Jones S, McMichael AJ, Rickinson AB, Callan MF. 1999. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J Immunol, 162 (3), pp. 1827-1835. | Show Abstract

EBV is a gammaherpesvirus that can establish both nonproductive (latent) and productive (lytic) infections within the cells of its host. Although T cell responses to EBV latent proteins have been well characterized, little is known about the importance of responses to lytic proteins in long term virus carriers. Here we have compared the frequencies of CD8+ T cells specific for EBV latent and lytic Ags in healthy virus carriers, using three techniques: limiting dilution analysis, enzyme-linked immunospot assay, and FACS staining with tetrameric MHC-peptide complexes. T cells specific for EBV lytic protein epitopes were readily detectable in all donors and were usually more abundant than those specific for latent epitopes. We infer that direct T cell control of viral replicative lesions is maintained in long term carriers of EBV and is an important component of the immune response to this virus. Estimates of CD8+ T cell frequencies varied considerably according to methodology; values obtained from MHC-peptide tetramer staining were, on the average, 4.4-fold higher than those obtained from enzyme-linked immunospot assays, which were, in turn, on the average, 5.3-fold higher than those obtained from limiting dilution analysis. Tetramer staining showed that as many as 5.5% circulating CD8+ T cells in a virus carrier were specific for a single EBV lytic protein epitope. Such values are much greater than previously imagined and illustrate how antigenic challenge from a persistent herpesvirus can influence the composition of the host's CD8+ T cell pool.

Dorrell L, Dong T, Ogg GS, Lister S, McAdam S, Rostron T, Conlon C, McMichael AJ, Rowland-Jones SL. 1999. Distinct recognition of non-clade B human immunodeficiency virus type 1 epitopes by cytotoxic T lymphocytes generated from donors infected in Africa. J Virol, 73 (2), pp. 1708-1714. | Show Abstract

We present detailed studies of human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses to clade A or C HIV type 1 in three donors infected in East Africa. We define several novel non-clade B CTL epitopes, including some restricted by HLA alleles common in Africans. Although cross-clade CTL recognition of these epitopes does occur, recognition can also be highly clade specific.

Braud VM, Allan DS, McMichael AJ. 1999. Functions of nonclassical MHC and non-MHC-encoded class I molecules. Curr Opin Immunol, 11 (1), pp. 100-108. | Show Abstract | Read more

Fascinating recent discoveries have focused attention on the nonclassical class I molecules. They can exert their function at most levels of the immune response, being part of both innate and adaptive immune systems. They not only have specialized antigen-presentation functions but also play important immunoregulatory roles: HLA-E regulates natural killer cells by interacting with CD94/NKG2 receptors; the MIC (MHC class I chain related) glycoproteins appear crucial to the activation of gammadelta T cells in the gastrointestinal epithelium; HLA-G may play a role in controlling the immune response to the fetus; and CD1 molecules are important in defense against bacterial infections, as well as in the development and regulation of a subset of NKT cells expressing a highly restricted TCR repertoire; however not all nonclassical class I molecules have an immunological function, as demonstrated by HFE which is implicated in iron metabolism.

Mongkolsapaya J, Jaye A, Callan MF, Magnusen AF, McMichael AJ, Whittle HC. 1999. Antigen-specific expansion of cytotoxic T lymphocytes in acute measles virus infection. J Virol, 73 (1), pp. 67-71. | Show Abstract

Skewing of the T-cell receptor repertoire of CD8(+) T cells has been shown in some persistent infections with viruses, such as human immunodeficiency virus, simian immunodeficiency virus, and Epstein-Barr virus. We have demonstrated that similar distortions also occur in nonpersistent measles virus infection. In addition, two of four children immunized with live, attenuated measles virus showed larger and more persistent CD8(+) T-cell expansions than their naturally infected counterparts. The expanded lymphocyte populations were monoclonal or oligoclonal and lysed target cells infected with recombinant vaccinia virus expressing measles virus protein. These results demonstrate that the expansions of CD8(+) T lymphocytes are antigen driven.

Goulder PJ, Rowland-Jones SL, McMichael AJ, Walker BD. 1999. Anti-HIV cellular immunity: recent advances towards vaccine design. AIDS, 13 Suppl A pp. S121-S136.

O'callaghan CA, Byford MF, Wyer JR, Willcox BE, Jakobsen BK, McMichael AJ, Bell JI. 1999. BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation. Anal Biochem, 266 (1), pp. 9-15. | Show Abstract | Read more

The enzyme BirA is a key reagent because of its ability to biotinylate proteins at a specific residue in a recognition sequence. We report a rapid, efficient, and economical method for the production, purification, and application of this enzyme. The method is easily scaled up and the protein produced is of high purity and can be stored for many months with retention of activity. We have used this enzyme to biotinylate the C termini of membrane proteins, allowing these proteins to be tetramerized by binding to streptavidin. Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface. These tetrameric proteins can be used to study protein receptor-ligand interactions at the cell surface, and site-specific biotinylation can be used to study proteins in vitro using a defined orientation.

Ogg GS, Jin X, Bonhoeffer S, Moss P, Nowak MA, Monard S, Segal JP, Cao Y et al. 1999. Decay kinetics of human immunodeficiency virus-specific effector cytotoxic T lymphocytes after combination antiretroviral therapy. J Virol, 73 (1), pp. 797-800. | Show Abstract

Little is known of the changes in human immunodeficiency virus type 1 (HIV-1)-specific effector cytotoxic T lymphocytes (CTL) after potent antiretroviral therapy. Using HLA/peptide tetrameric complexes, we show that after starting treatment, there are early rapid fluctuations in the HIV-1-specific CTL response which last 1 to 2 weeks. These fluctuations are followed by an exponential decay (median half-life, 45 days) of HIV-1-specific CTL which continues while viremia remains undetectable. These data have implications for the immunological control of drug-resistant virus.

Braud VM, McMichael AJ. 1999. Regulation of NK cell functions through interaction of the CD94/NKG2 receptors with the nonclassical class I molecule HLA-E. Curr Top Microbiol Immunol, 244 pp. 85-95.

Dyer WB, Ogg GS, Demoitie MA, Jin X, Geczy AF, Rowland-Jones SL, McMichael AJ, Nixon DF, Sullivan JS. 1999. Strong human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte activity in Sydney Blood Bank Cohort patients infected with nef-defective HIV type 1. J Virol, 73 (1), pp. 436-443. | Show Abstract

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with natural nef and nef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.

Callan MF, McMichael AJ. 1999. T cell receptor usage in infectious disease. Springer Semin Immunopathol, 21 (1), pp. 37-54. | Read more

Callan MF, Annels N, Steven N, Tan L, Wilson J, McMichael AJ, Rickinson AB. 1998. T cell selection during the evolution of CD8+ T cell memory in vivo. Eur J Immunol, 28 (12), pp. 4382-4390. | Show Abstract | Read more

Memory T cell responses are frequently highly restricted in terms of receptor usage. How and when such clonotypic dominance is established remains poorly understood. Here we have investigated the evolution of the T cell responses to an epitope from Epstein-Barr virus (EBV), (FLRGRAYGL), by analyzing TCR use of clones specific for this epitope, derived from peripheral blood mononuclear cells taken from individuals early during primary EBV infection and up to 3 years later. We show that, in a given individual, particular T cell clonotypes are selected early during the primary response to this epitope and that the same clonotypes dominate the late memory response. In one individual direct analysis of HLA-B8-restricted FLRGRAYGL-specific T cells, isolated from peripheral blood lymphocytes taken during primary EBV infection using a tetrameric MHC-peptide complex, confirmed the early selection of the dominant clonotypes.

Tan R, Wilson JD, Rostron T, Peto T, McMichael AJ, Rowland-Jones SL. 1998. Prolonged CD8+ T-cell expansions after adoptive transfer of syngeneic lymphocytes between HIV-discordant identical twins. AIDS, 12 (16), pp. 2240-2241.

Rowland-Jones SL, Dong T, Fowke KR, Kimani J, Krausa P, Newell H, Blanchard T, Ariyoshi K et al. 1998. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J Clin Invest, 102 (9), pp. 1758-1765. | Show Abstract | Read more

Many people who remain persistently seronegative despite frequent HIV exposure have HIV-specific immune responses. The study of these may provide information about mechanisms of natural protective immunity to HIV-1. We describe the specificity of cytotoxic T lymphocyte responses to HIV in seronegative prostitutes in Nairobi who are apparently resistant to HIV infection. These women have had frequent exposure to a range of African HIV-1 variants, primarily clades A, C, and D, for up to 12 yr without becoming infected. Nearly half of them have CTL directed towards epitopes previously defined for B clade virus, which are largely conserved in the A and D clade sequences. Stronger responses are frequently elicited using the A or D clade version of an epitope to stimulate CTL, suggesting that they were originally primed by exposure to these virus strains. CTL responses have been defined to novel epitopes presented by HLA class I molecules associated with resistance to infection in the cohort, HLA-A*6802 and HLA-B18. Estimates using a modified interferon-gamma Elispot assay indicate a circulating frequency of CTL to individual epitopes of between 1:3,200 and 1:50,000. Thus, HIV-specific immune responses-particularly cross-clade CTL activity- may be responsible for protection against persistent HIV infection in these African women.

Maini MK, Ogg GS, Boni C, Pilli M, Ferrari C, McMichael AJ, Williams R, Vergani D, Bertoletti A. 1998. Direct visualisation of hepatitis B virus (HBV)-specific cytotoxic T cells with an human leucocyte antigen (HLA) class I peptide tetrameric complex allows a new insight into their actual frequency. HEPATOLOGY, 28 (4), pp. 485A-485A.

Bowness P, Allen RA, McMichael AJ. 1998. HLA B27 can form heavy chain homodimers in vivo and in vivo, possible role of cys 67. ARTHRITIS AND RHEUMATISM, 41 (9), pp. S355-S355.

Bowness P, Allen RL, Barclay DN, Jones EY, McMichael AJ. 1998. Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex. Eur J Immunol, 28 (9), pp. 2704-2713. | Show Abstract | Read more

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.

Wilson JD, Ogg GS, Allen RL, Goulder PJ, Kelleher A, Sewell AK, O'Callaghan CA, Rowland-Jones SL, Callan MF, McMichael AJ. 1998. Oligoclonal expansions of CD8(+) T cells in chronic HIV infection are antigen specific. J Exp Med, 188 (4), pp. 785-790. | Show Abstract | Read more

Acute HIV infection is associated with a vigorous immune response characterized by the proliferation of selected T cell receptor V beta (BV)-expressing CD8(+) T cells. These 'expansions', which are commonly detected in the peripheral blood, can persist during chronic HIV infection and may result in the dominance of particular clones. Such clonal populations are most consistent with antigen-driven expansions of CD8(+) T cells. However, due to the difficulties in studying antigen-specific T cells in vivo, it has been hard to prove that oligoclonal BV expansions are actually HIV specific. The use of tetrameric major histocompatibility complex-peptide complexes has recently enabled direct visualization of antigen-specific T cells ex vivo but has not provided information on their clonal composition. We have now made use of these tetrameric complexes in conjunction with anti-BV chain-specific monoclonal antibodies and analysis of cytotoxic T lymphocyte lines/clones to show that chronically clonally expanded CD8(+) T cells are HIV specific in vivo.

Ogg GS, McMichael AJ. 1998. HLA-peptide tetrameric complexes. Curr Opin Immunol, 10 (4), pp. 393-396. | Show Abstract | Read more

HLA-peptide tetrameric complexes allow the direct ex vivo visualisation of antigen-specific CD8+ T cells and natural killer cells by flow cytometry. Quantitation, phenotypic analysis and isolation of tetramer-binding cells has considerably extended our understanding of cytotoxic T lymphocyte and natural killer cell function.

McMichael AJ. 1998. The original sin of killer T cells. Nature, 394 (6692), pp. 421-422. | Read more

McMichael AJ, O'Callaghan CA. 1998. A new look at T cells. J Exp Med, 187 (9), pp. 1367-1371. | Read more

Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, O'Callaghan CA, Steven N, McMichael AJ, Rickinson AB. 1998. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo. J Exp Med, 187 (9), pp. 1395-1402. | Show Abstract | Read more

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex-peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8+ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8+ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.

O'Callaghan CA, Tormo J, Willcox BE, Blundell CD, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E. Protein Sci, 7 (5), pp. 1264-1266. | Show Abstract | Read more

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

Hall FC, Thomson K, Procter J, McMichael AJ, Wordsworth BP. 1998. TCR beta spectratyping in RA: evidence of clonal expansions in peripheral blood lymphocytes. Ann Rheum Dis, 57 (5), pp. 319-322. | Show Abstract | Read more

OBJECTIVE: To compare the TCR beta repertoire of peripheral blood CD8 enriched (CD8+) and depleted (CD8-) T cells in rheumatoid arthritis (RA) patients and controls using CDR3 length analysis (spectratyping). METHODS: CD8+ and CD8- T cells were separated from 14 RA patients and 12 controls, using magnetic beads coated with anti-CD8 monoclonal antibodies. cDNA was prepared as the template for amplification with 22 V beta-C beta primer pairs. The products were resolved by electrophoresis in an ABI373 sequencer using GENESCAN software. Expansions were identified as dominant CDR3 lengths, where the area underlying the corresponding peak exceeded the sum of the areas of the two adjacent peaks. This method was validated by sequencing 10 samples displaying dominant peaks. The expansion frequencies in RA patients and controls were compared using the chi 2 test statistic. RESULTS: Dominant peaks were evident in several V beta families. They were more frequent in RA patients in both the CD8+ subset (RA normalised frequency 10.6; control normalised frequency 8.0; p = 0.03) and the CD8- subset (RA normalised frequency 2.9; control normalised frequency 1.5; p = 0.02). Sequencing of 10 samples exhibiting dominant peaks revealed an unequivocal clonal expansion in nine (90%). CONCLUSIONS: RA patients exhibited a significantly increased frequency of T cell expansions both in the CD8+ and CD8- subsets. This phenomenon may reflect the proliferation of autoreactive cells, a nonspecific expansion of memory T cells in response to pro-inflammatory cytokines or a defect of T cell regulation that predates the onset of RA and may itself predipose to disease.

Tafuro S, Hourigan C, McMichael AJ, Bell J, Moss PAH. 1998. Use of an internal ribosomal entry site to express two genes from a dicistronic construct after retroviral infection of cytotoxic T cells. BRITISH JOURNAL OF HAEMATOLOGY, 101 pp. 98-98.

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Beral V, Babiker A, Brettle R, Carne C, Darbyshire JH, Evans BG, Gill ON, Gilson RJC et al. 1998. The AIDS incubation period in the UK estimated from a national register of HIV seroconverters AIDS, 12 (6), pp. 659-667.

Wilson JD, Cranage M, Cook N, Leech S, McMichael AJ, Callan MF. 1998. Evidence for the persistence of monoclonal expansions of CD8+ T cells following primary simian immunodeficiency virus infection. Eur J Immunol, 28 (4), pp. 1172-1180. | Show Abstract | Read more

A longitudinal study of the CD8+ TCR variable (Vbeta) chain repertoire was performed in rhesus macaques experimentally infected with simian immunodeficiency virus (SIV) using both TCR Vbeta chain-specific monoclonal antibodies and TCR beta chain CDR3 length analysis. Expansions of subpopulations of CD8+ T cells were detected during the acute phase of SIV infection. In all monkeys studied, monoclonal expansions persisted for at least 18 months and increasingly dominated the repertoire of CD8+ T cells expressing the relevant Vbeta chain. This study shows that persistent CD8+ T cell expansions develop in response to a virus infection. This is important not only for our understanding of the T cell response to viruses but also for understanding the factors that determine the normal CD8+ TCR repertoire.

Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y et al. 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science, 279 (5359), pp. 2103-2106. | Show Abstract | Read more

Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.

O'Callaghan CA, Tormo J, Willcox BE, Braud VM, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Mol Cell, 1 (4), pp. 531-541. | Show Abstract | Read more

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Braud VM, Allan DS, O'Callaghan CA, Söderström K, D'Andrea A, Ogg GS, Lazetic S, Young NT et al. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 391 (6669), pp. 795-799. | Show Abstract | Read more

The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.

Braud VM, McMichael AJ, Cerundolo V. 1998. Differential processing of influenza nucleoprotein in human and mouse cells. Eur J Immunol, 28 (2), pp. 625-635. | Show Abstract | Read more

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265-273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265-273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.

Mongkolsapaya J, Cowper AE, Xu XN, Morris G, McMichael AJ, Bell JI, Screaton GR. 1998. Lymphocyte inhibitor of TRAIL (TNF-related apoptosis-inducing ligand): a new receptor protecting lymphocytes from the death ligand TRAIL. J Immunol, 160 (1), pp. 3-6. | Show Abstract

Apoptosis can be triggered by the engagement of cell surface receptors by their ligands. A growing number of receptors belonging to the TNF receptor family have been identified that contain a conserved cytoplasmic death domain. These include Fas, TNF-R1, lymphocyte-associated receptor of death (LARD), DR4, and TNF-related apoptosis-inducing ligand receptor inducer of cell killing-2 (TRICK2). The latter two are receptors for the cytotoxic ligand TNF-related apoptosis-inducing ligand (TRAIL), and one of the paradoxes raised by the cloning of these molecules was why do most cells not die upon contact with the widely expressed TRAIL molecule? This is a particular problem for lymphocytes that express DR4 and TRICK2 and are in constant circulation through TRAIL-expressing tissues. We have cloned LIT (lymphocyte inhibitor of TRAIL), which lacks a death domain. LIT is expressed predominantly on PBL, where it can competitively inhibit TRAIL-induced apoptosis through DR4/TRICK2, and may function to modulate lymphocyte sensitivity to TRAIL.

Braud VM, Allan DS, Wilson D, McMichael AJ. 1998. TAP- and tapasin-dependent HLA-E surface expression correlates with the binding of an MHC class I leader peptide. Curr Biol, 8 (1), pp. 1-10. | Show Abstract | Read more

BACKGROUND: The human major histocompatibility complex (MHC) class lb molecule HLA-E is transcribed in most tissues but little is known about its localisation within the cell. We have recently shown that HLA-E binds signal-sequence-derived peptides from human MHC class I molecules in vitro. RESULTS: Using a newly characterised antibody recognising HLA-E, we show that HLA-E is expressed at the cell surface. We demonstrate that HLA-E surface expression is correlated with the presence of MHC class I molecules which provide suitable leader sequence peptides capable of binding to HLA-E. Further studies on the interaction of HLA-E with molecules in the endoplasmic reticulum revealed that HLA-E associates with the transporter associated with antigen processing (TAP) and calreticulin, and that HLA-E expression is TAP-dependent and tapasin-dependent. In addition, HLA-E dissociates from TAP upon binding of MHC class I leader sequence peptides. CONCLUSION: These experiments establish that surface expression of HLA-E is regulated by the binding of a restricted pool of peptides from the leader sequence of MHC class I molecules. The correlation between HLA-E and MHC class I surface expression might be relevant to the function of HLA-E. Our results also show that, although these HLA-E binding peptides are derived from signal sequences, they may be released back into the cytosol and subsequently translocated by the TAP complex and loaded onto HLA-E molecules.

Lalvani A, Dong T, Ogg G, Patham AA, Newell H, Hill AV, McMichael AJ, Rowland-Jones S. 1997. Optimization of a peptide-based protocol employing IL-7 for in vitro restimulation of human cytotoxic T lymphocyte precursors. J Immunol Methods, 210 (1), pp. 65-77. | Show Abstract | Read more

A variety of different methods for the in vitro restimulation of human cytotoxic T lymphocyte (CTL) precursors (CTLp) are in use. Our aim was to enhance the detection of circulating human CTLp in peripheral blood. We have developed a standardized and highly efficient method for restimulating CTLp. Synthetic peptides were used to restimulate cognate CTLp from peripheral blood mononuclear cells (PBMC), and effector CTL capable of lysing peptide-pulsed and virus infected targets were generated. The effects of several parameters on CTL specific for influenza A, EBV and HIV-1 were evaluated, and the optimum peptide concentration for CTL generation was established. Supplementation of initial cultures with IL-7 greatly enhanced peptide-specific lytic activity for all peptides tested and the dose-response relationship for IL-7 was delineated. A novel technique using peptide-MHC class I molecule tetramers to stain T cells bearing cognate T cell receptors permitted enumeration of antigen-specific CD8 + CTL during in vitro restimulation; IL-7 supplementation selectively expanded the population of peptide-specific CD8 + CTL. Importantly, this protocol, whilst enhancing the restimulation and lytic activity of secondary CTL, does not induce primary CTL in vitro. The improved efficiency with which CTL are generated in this system substantially enhances the sensitivity of CTL culture and the 51Cr release assay to detect low levels of CTL activity.

Goulder PJ, Edwards A, Phillips RE, McMichael AJ. 1997. Identification of a novel HLA-A24-restricted cytotoxic T-lymphocyte epitope within HIV-1 Nef. AIDS, 11 (15), pp. 1883-1884. | Read more

Goulder PJ, Bunce M, Luzzi G, Phillips RE, McMichael AJ. 1997. Potential underestimation of HLA-C-restricted cytotoxic T-lymphocyte responses. AIDS, 11 (15), pp. 1884-1886. | Read more

Lalvani A, Brookes R, Hambleton S, Britton WJ, Hill AV, McMichael AJ. 1997. Rapid effector function in CD8+ memory T cells. J Exp Med, 186 (6), pp. 859-865. | Show Abstract | Read more

The nature of the CD8+ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8+ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon gamma (IFN-gamma) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8+ T cells displayed IFN-gamma release within 6 h of antigen contact, identifying a population of memory CD8+ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8+ effector T cells specific for six different MHC class I-restricted influenza virus epitopes. Enumeration of these CD8+ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8+ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.

Allen RL, Gillespie GM, Hall F, Edmonds S, Hall MA, Wordsworth BP, McMichael AJ, Bowness P. 1997. Multiple T cell expansions are found in the blood and synovial fluid of patients with reactive arthritis. J Rheumatol, 24 (9), pp. 1750-1757. | Show Abstract

OBJECTIVE: To look for evidence of T lymphocyte expansions in the blood and synovial fluid (SF) of patients with reactive arthritis (ReA). METHODS: Paired peripheral blood and synovial samples from 10 patients with ReA were studied by dual color flow cytometry using T cell receptor (TCR) V beta specific and CD4 or CD8 specific antibodies. Two synovial CD8 expansions were studied by 3 color flow cytometry. Peripheral blood samples from 13 healthy, age matched individuals were studied as controls. RESULTS: Statistically significant expansions were observed in all patients, occurring in blood and SF CD4 and CD8 compartments, but were most common in the synovial CD8 compartment. Expansions studied in further detail displayed an activated "memory" phenotype. A synovial BV22S1/CD8 expansion was seen in 5/6 patients with sexually acquired ReA. CONCLUSION: Multiple T lymphocyte expansions are found in both the blood and SF of patients with ReA. Expansions were most commonly found in the synovial CD8 compartment, where they appeared to express both activation and memory markers. This indicates that T lymphocytes (and in particular cytotoxic T cells) may play a pathogenic role in ReA. These findings are consistent with either an antigen or a superantigen driven response.

Screaton GR, Mongkolsapaya J, Xu XN, Cowper AE, McMichael AJ, Bell JI. 1997. TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL. Curr Biol, 7 (9), pp. 693-696. | Show Abstract | Read more

A subset of the tumour necrosis factor (TNF) receptor family contain a conserved intracellular motif, the death domain. Engagement of these receptors by their respective ligands initiates a signalling cascade that rapidly leads to cell death by apoptosis. We have cloned a new member of this family, TRICK2, the TRAIL (TNF-related apoptosis-inducing ligand) receptor inducer of cell killing 2. TRICK2 is expressed in a number of cell types, and to particularly high levels in lymphocytes and spleen. Two isoforms of the TRICK2 mRNA are generated by alternative pre-mRNA splicing and differ by a 29 amino-acid extension to the extracellular domain. Overexpression of TRICK2 rapidly induced apoptosis in 293T cells; this induction was dependent upon the presence of the death domain of TRICK2. Using a soluble molecule containing the TRICK2 extracellular domain, we demonstrated that TRICK2, like DR4 [1], is a receptor for TRAIL/APO-2L [2,3] and could inhibit TRAIL-induced killing of lymphocyte lines, such as the Jurkat T-cell line. TRAIL is upregulated upon lymphocyte activation, as is the intensively studied ligand for Fas, FasL [4]. TRAIL and its receptors might therefore provide another system for the regulation of lymphocyte selection and proliferation, as well as providing an additional weapon in the armoury of cytotoxic lymphocytes.

Xu XN, Screaton GR, Gotch FM, Dong T, Tan R, Almond N, Walker B, Stebbings R et al. 1997. Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells. J Exp Med, 186 (1), pp. 7-16. | Show Abstract | Read more

Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four-amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4(+) and CD8(+) populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.

Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. 1997. Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2. Nature, 387 (6633), pp. 630-634. | Show Abstract | Read more

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8alpha(alpha) or the heterodimer alpha beta stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 A resolution of a complex between CD8alpha(alpha) and the human MHC molecule HLA-A2, which is associated with peptide. CD8alpha(alpha) binds one HLA-A2/peptide molecule, interfacing with the alpha2 and alpha3 domains of HLA-A2 and also contacting beta2-microglobulin. A flexible loop of the alpha3 domain (residues 223-229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the alpha3 domain is different from that in uncomplexed HLA-A2, being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in alpha3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

Goulder PJ, Reid SW, Price DA, O'Callaghan CA, McMichael AJ, Phillips RE, Jones EY. 1997. Combined structural and immunological refinement of HIV-1 HLA-B8-restricted cytotoxic T lymphocyte epitopes. Eur J Immunol, 27 (6), pp. 1515-1521. | Show Abstract | Read more

This study demonstrates that use of structural information improves the definition and optimization of cytotoxic T lymphocyte (CTL) epitopes. Epitope optimization usually requires numerous truncated peptides or a reverse immunogenetic approach, where the peptide binding motif is used to predict epitopes. These binding motifs do not reliably predict all peptides which are CTL epitopes. Comparison of 24 peptides eluted from HLA-B8 with 10 HLA-B8-restricted defined CTL epitopes demonstrated that known epitopes varied considerably at anchor positions. We used structural information based on determination of the crystal structure of the HLA-B8-GGKKKYKL complex to reassess previously described CTL epitopes, to predict new epitopes, and to predict the consequences of naturally occurring variation within epitopes. These predictions were confirmed by cytotoxicity and binding assays. Use of combined structural and immunological data more accurately defines the true peptide-binding motif of a restriction element than eluted peptide data allows.

Goulder PJ, Edwards A, Phillips RE, McMichael AJ. 1997. Identification of a novel HLA-B*3501-restricted cytotoxic T lymphocyte epitope using overlapping peptides. AIDS, 11 (7), pp. 930-932.

Screaton GR, Xu XN, Olsen AL, Cowper AE, Tan R, McMichael AJ, Bell JI. 1997. LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing. Proc Natl Acad Sci U S A, 94 (9), pp. 4615-4619. | Show Abstract | Read more

Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the "death domain." We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.

Goulder PJ, Sewell AK, Lalloo DG, Price DA, Whelan JA, Evans J, Taylor GP, Luzzi G, Giangrande P, Phillips RE, McMichael AJ. 1997. Patterns of immunodominance in HIV-1-specific cytotoxic T lymphocyte responses in two human histocompatibility leukocyte antigens (HLA)-identical siblings with HLA-A*0201 are influenced by epitope mutation. J Exp Med, 185 (8), pp. 1423-1433. | Show Abstract | Read more

Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201-restricted SLYNTVATL and HLA-A3-restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201-restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response.

Vessey SJ, Barouch DH, McAdam SN, Tussey LG, Davenport MA, O'Callaghan CA, Bell JI, McMichael AJ, Jakobsen BK. 1997. Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide. Eur J Immunol, 27 (4), pp. 879-885. | Show Abstract | Read more

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.

Goulder PJ, Edwards A, Phillips RE, McMichael AJ. 1997. Identification of a novel HLA-B*2705-restricted cytotoxic T-lymphocyte epitope within a conserved region of HIV-1 Nef. AIDS, 11 (4), pp. 536-538.

Goulder PJ, Phillips RE, Colbert RA, McAdam S, Ogg G, Nowak MA, Giangrande P, Luzzi G et al. 1997. Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat Med, 3 (2), pp. 212-217. | Show Abstract | Read more

The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial. Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops. It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape. The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection. We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope. In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability. CTL escape may play an important role in the pathogenesis of HIV infection.

Cited:

322

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Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. 1997. Crystal structure of the complex between CD8αα human and HLA-A2 Nature, 387 (6633), pp. 630-634. | Show Abstract | Read more

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8αα or the heterodimer αβ stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class l/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 Å resolution of a complex between CD8αα and the human MHC molecule HLA-A2, which is associated with peptide. CD8αα binds one HLA-A2/peptide molecule, interfacing with the α2 and α3 domains of HLA-A2 and also contacting β2- microglobulin. A flexible loop of the α3 domain (residues 223-229) is damped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the α3 domain is different from that in uncomplexed HLA-A2 (refs 3, 4), being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in α3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is dearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

McMichael AJ, Phillips RE. 1997. Escape of human immunodeficiency virus from immune control. Annu Rev Immunol, 15 (1), pp. 271-296. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTL) play a crucial role in the attempt to control infection with human immunodeficiency virus (HIV). Variation in epitopes recognized by CTL is common and frequently offers potential escape routes for mutant virus. Proof of escape, however, requires demonstration of increased frequency of virus particles or provirus that carry the escape sequence. There are now several recorded examples of virus variants that escape from CTL and are then selected. Most dramatic are those in which the CTL response has been dominated by CTL recognizing a single epitope that has suddenly changed, resulting in escape to fixation. This has been seen both early and late in the infection, leaving no doubt that escape occurs. Such escape is likely to be favored when the antiviral CTL response is oligoclonal and focused on a small number of immunodominant epitopes. The heterogeneous CTL response seen in many HIV-infected patients may result from successive waves of virus escape followed by new CTL responses specific for subdominant epitopes. Mutant virus can escape by several different routes, including failure of the mutated peptide to bind to the presenting HLA molecule and altered interactions with T cell receptors (TCR), including antagonism.

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.

Goulder PJ, Bunce M, Krausa P, McIntyre K, Crowley S, Morgan B, Edwards A, Giangrande P, Phillips RE, McMichael AJ. 1996. Novel, cross-restricted, conserved, and immunodominant cytotoxic T lymphocyte epitopes in slow progressors in HIV type 1 infection. AIDS Res Hum Retroviruses, 12 (18), pp. 1691-1698. | Show Abstract | Read more

HIV-specific cytotoxic T lymphocytes (CTLs) play an important role in the immune response to HIV infection. Long-term nonprogressors (LTNPs) or slow progressors (SPs) in HIV infection may make qualitatively different CTL responses compared to those generated by seropositive individuals who progress to disease at a faster rate. The class I molecule HLA-B*57 has been identified as one restriction element overrepresented in SP groups studied, and, together with the closely related molecule HLA-B*58, occurs commonly in ethnic groups where HIV is most prevalent. In this study, we have identified five new HLA-B*57-restricted CTL epitopes recognized by SP donors, one of which is also HLA-B*5801 restricted. These HLA-B*57-restricted responses represent the dominant HIV-specific CTL response in each of the SP donors tested. These and other such epitopes may be an important component in future vaccine design.

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.

Nowak MA, Anderson RM, Boerlijst MC, Bonhoeffer S, May RM, McMichael AJ. 1996. HIV-1 evolution and disease progression. Science, 274 (5289), pp. 1008-1011. | Read more

Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science, 274 (5284), pp. 94-96. | Show Abstract | Read more

Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general, tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific for infectious agents, tumors, and autoantigens.

Mongkolsapaya J, Callan MFC, McMichael AJ. 1996. Differentiation and activation antigens on CD8 T cells TISSUE ANTIGENS, 48 (4-II), pp. TC406-TC406.

Callan MF, Steven N, Krausa P, Wilson JD, Moss PA, Gillespie GM, Bell JI, Rickinson AB, McMichael AJ. 1996. Large clonal expansions of CD8+ T cells in acute infectious mononucleosis. Nat Med, 2 (8), pp. 906-911. | Show Abstract | Read more

Primary infection with Epstein-Barr virus often results in the clinical syndrome of acute infectious mononucleosis (glandular fever). This illness is characterized by a striking lymphocytosis, the nature of which has been controversial. We show that large monoclonal or oligoclonal populations of CD8+ T cells account for a significant proportion of the lymphocytosis and provide molecular evidence that these populations have been driven by antigen. The results suggest that the selective and massive expansion of a few dominant clones of CD8+ T cells is an important feature of the primary response to this virus.

McMichael AJ. 1996. HIV. The immune response. Curr Opin Immunol, 8 (4), pp. 537-539. | Read more

Gao XM, Wordsworth P, McMichael AJ, Kyaw MM, Seifert M, Rees D, Dougan G. 1996. Homocysteine modification of HLA antigens and its immunological consequences. Eur J Immunol, 26 (7), pp. 1443-1450. | Show Abstract | Read more

Homocysteine-treated cells can be specifically lysed by cytotoxic T lymphocytes (CTL) identifiable in patients with ankylosing spondylitis and reactive arthritis. Sensitization of target cells involves disulfide bonding and the interaction between homocysteine and HLA antigens occurs in a pre-Golgi compartment in the cells. Salmonella-infected B cells are also lysed by homocysteine-specific CTL, suggesting that intracellular invading microorganisms may provide homocysteine which would gain access to the newly synthesized intracellular HLA molecules and modify them inside the cells. Two different mechanisms for homocysteine modification of HLA antigens are proposed: homocysteine could bind directly to the unpaired cysteine residues in HLA antigens, or it could bind indirectly to HLA antigens through cysteine-containing peptides bound to them. Thus, HLA antigens containing unpaired cysteine residues (e.g. HLA B27) could be modified by homocysteine directly or indirectly, while HLA antigens without unpaired cysteine residues (e.g. HLA A68) could only be modified indirectly. The results are discussed in relation to the potential involvement of homocysteine-specific CTL in ankylosing spondylitis and reactive arthritis, both of which are related to bacterial infections, associated with HLA B27, and considered to be autoimmune diseases.

McClure MO, Goulder PJ, Ogg G, McMichael AJ, Weber JN. 1996. HIV clearance in infants. Lancet, 347 (9008), pp. 1122. | Read more

Reid SW, Smith KJ, Jakobsen BK, O'Callaghan CA, Reyburn H, Harlos K, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1996. Production and crystallization of MHC class I B allele single peptide complexes. FEBS Lett, 383 (1-2), pp. 119-123. | Show Abstract | Read more

Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.

Smith KJ, Reid SW, Stuart DI, McMichael AJ, Jones EY, Bell JI. 1996. An altered position of the alpha 2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity, 4 (3), pp. 203-213. | Show Abstract | Read more

The crystal structure of the human major histocompatibility complex class I B allele HLA B*3501 complexed with the 8-mer peptide epitope HIV1 Nef 75-82 (VPLRPMTY) has been determined at 2.0 angstrom resolution. Comparison with the crystal structure of the closely related allele HLA B*5301 reveals the structural basis for the tyrosine specificity of the B*3501 F pocket. The structure also reveals a novel conformation of the 8-mer peptide within the binding groove. The positions of the peptide N and C termini are nonstandard, but the classic pattern of hydrogen bonding to nonpolymorphic MHC class I residues is maintained, at the N terminus by addition of a water molecule, and at the C terminus by a substantial shift in the alpha 2 helix.

Smith KJ, Reid SW, Harlos K, McMichael AJ, Stuart DI, Bell JI, Jones EY. 1996. Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53. Immunity, 4 (3), pp. 215-228. | Show Abstract | Read more

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.

Bangham CRM, McMichael AJ. 1996. Presentation of viral antigens to cytotoxic T cells - Introduction SEMINARS IN VIROLOGY, 7 (1), pp. 1-2. | Read more

Toshitani K, Braud V, Browning MJ, Murray N, McMichael AJ, Bodmer WF. 1996. Expression of a single-chain HLA class I molecule in a human cell line: presentation of exogenous peptide and processed antigen to cytotoxic T lymphocytes. Proc Natl Acad Sci U S A, 93 (1), pp. 236-240. | Show Abstract | Read more

We have synthesized a recombinant gene encoding a single-chain HLA-A2/beta 2-microglobulin (beta 2m) molecule by linking beta 2m through its carboxyl terminus via a short peptide spacer to HLA-A2 (A*0201). This gene has been expressed in the beta 2m-deficient colorectal tumor cell line DLD-1. Transfection of this cell with the single-chain construct was associated with conformationally correct cell surface expression of a class I molecule of appropriate molecular mass. The single-chain HLA class I molecule presented either exogenously added peptide or (after interferon-gamma treatment) endogenously processed antigen to an influenza A matrix-specific, HLA-A2-restricted cytotoxic T-lymphocyte line. The need for interferon gamma for the processing and presentation of endogenous antigen suggests that DLD-1 has an antigen-processing defect that can be up-regulated, a feature that may be found in other carcinomas. Our data indicate that single-chain HLA class I constructs can form functional class I molecules capable of presenting endogenously processed antigens. Such molecules should be of use for functional studies, as well as providing potential anticancer immunotherapeutic agents or vaccines.

Gao XM, McMichael AJ. 1996. Cytotoxic T lymphocytes specific for murine type II collagen do not trigger arthritis in B10 mice. Clin Exp Immunol, 103 (1), pp. 89-93. | Show Abstract | Read more

To investigate the role of cytotoxic T lymphocytes (CTL) in arthritis, we set out to induce CTL specific for murine type II collagen (mCII) in a mouse model. The primary protein sequence of the murine pro-alpha 1(II) was screened for fragments bearing H-2 Db or Kb binding motifs. Six fragments were identified and the corresponding peptides synthesized. One of these peptides, peptide P201 (amino acid 199-208 in the C-propeptide of the murine pro-alpha 1(II), was found to be a strong binder to H-2 Db. When used to treat RMA-S cells at 26 degrees C, peptide P201 induced a four-fold increase of surface expression of H-2 Db. Administration of the P201-treated RMA-S cells into B10 mice (H-2b) induced strong CTL responses against the immunizing collagen peptide. Despite the high frequencies of mCII-specific CTL precursors in the periphery, however, the immunized mice showed no sign of arthritis up to 16 weeks after immunization. Implications of these data for autoimmunity and arthritis are discussed.

Haurum JS, Tan L, Arsequell G, Frodsham P, Lellouch AC, Moss PA, Dwek RA, McMichael AJ, Elliott T. 1995. Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition. Eur J Immunol, 25 (12), pp. 3270-3276. | Show Abstract | Read more

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2Db using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind Db. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to Db. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the Db pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited Db-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all Db-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, Db-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to Db, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.

BOWNESS P, ALLEN R, MCMICHAEL A, EDMUNDS S, HALL M, BOWMAN S, WORDSWORTH B. 1995. HLA-B27-RESTRICTED PEPTIDE PRESENTATION TO CYTOTOXIC T-CELLS IN REACTIVE ARTHRITIS ARTHRITIS AND RHEUMATISM, 38 (9), pp. 174-174.

Nowak MA, McMichael AJ. 1995. How HIV defeats the immune system. Sci Am, 273 (2), pp. 58-65. | Read more

Gerth UC, Moss PA, Bell JI, McMichael AJ. 1995. T-cell receptor usage of major histocompatibility complex class I restricted peptide-specific T-lymphocytes. Ann N Y Acad Sci, 756 (1 T-Cell Recept), pp. 12-18. | Read more

Tussey LG, Rowland-Jones S, Zheng TS, Androlewicz MJ, Cresswell P, Frelinger JA, McMichael AJ. 1995. Different MHC class I alleles compete for presentation of overlapping viral epitopes. Immunity, 3 (1), pp. 65-77. | Show Abstract | Read more

We previously identified an HLA-B8+ donor, NW, whose lymphoblastoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-B2702-restricted influenza-specific CTL response to an epitope in the nucleoprotein that overlaps the B8-restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the B8-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B2702 epitope. The other is that B8 and B2702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-B8.

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.

Moss PA, Rowland-Jones SL, Frodsham PM, McAdam S, Giangrande P, McMichael AJ, Bell JI. 1995. Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors. Proc Natl Acad Sci U S A, 92 (13), pp. 5773-5777. | Show Abstract | Read more

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

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.

Hill AB, Lee SP, Haurum JS, Murray N, Yao QY, Rowe M, Signoret N, Rickinson AB, McMichael AJ. 1995. Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised. J Exp Med, 181 (6), pp. 2221-2228. | Show Abstract | Read more

We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release.

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HILL A, LEE S, HAURUM J, MURRAY N, YAO Q, ROWE M, SIGNORET N, RICKINSON A, MCMICHAEL A. 1995. CLASS-I MAJOR HISTOCOMPATIBILITY COMPLEX-RESTRICTED CYTOTOXIC T-LYMPHOCYTES SPECIFIC FOR EPSTEIN-BARR-VIRUS (EBV) NUCLEAR ANTIGENS FAIL TO LYSE THE EBV-TRANSFORMED B-LYMPHOBLASTOID CELL-LINES AGAINST WHICH THEY WERE RAISED JOURNAL OF EXPERIMENTAL MEDICINE, 181 (6), pp. 2221-2228. | Read more

Callan MF, Reyburn HT, Bowness P, Rowland-Jones S, Bell JI, McMichael AJ. 1995. Selection of T cell receptor variable gene-encoded amino acids on the third binding site loop: a factor influencing variable chain selection in a T cell response. Eur J Immunol, 25 (6), pp. 1529-1534. | Show Abstract | Read more

The 3' end of the T cell receptor V beta 7.1 gene contains the five nucleotides CAAGA between the broadly conserved consensus sequence of nucleotides TGC/T GCC AGC AGC (which encode cysteine, alanine, serine and serine at positions 92-95 of the beta chain) and the heptamer that signals rearrangement. These nucleotides are frequently preserved during gene rearrangement, resulting in the common presence of glutamine at position 96 and of aspartate or glutamate at position 97 of the V beta 7.1 chain CDR3 loop in peripheral blood lymphocytes. There is selection of V beta 7.1 and of the V beta 7.1 gene-encoded glutamate at position 97 of the beta chain CDR3 loop in the cytotoxic T lymphocyte response to the HLA B2705-restricted influenza A nucleoprotein epitope SRYWAIRTR. Our results indicate that selection of V beta 7.1 gene-encoded amino acid residues on CDR3 loops may be one factor driving selection of V beta 7.1 in this response.

Krausa P, Brywka M, Savage D, Hui KM, Bunce M, Ngai JL, Teo DL, Ong YW, Barouch D, Allsop CE. 1995. Genetic polymorphism within HLA-A*02: significant allelic variation revealed in different populations. Tissue Antigens, 45 (4), pp. 223-231. | Show Abstract | Read more

HLA-A2 is present at high frequency in most populations, as identified by serological and biochemical means. The value of these methods is limited by their failure to discriminate between the products of the 14 known allelic HLA-A*02 variants. The great majority of genetic polymorphism which defines the allelic variants is found in exons 2 and 3 of the A*02 genes. These exons encode the alpha-1 and alpha-2 domains of the HLA Class I molecules, and variation within the genes may influence the peptide binding specificity of the gene products of each allele. Failure to accurately assign the allelic types has implications in transplantation, in interpretation of cellular assays and in the understanding of HLA disease associations. We have developed a method for determining the 14 known alleles of HLA-A*02 by use of ARMS-PCR to determine the degree of variation of HLA-A*02 alleles in 3 different population groups. Considerable variation was found in the relative frequencies of particular A*02 alleles between Caucasian, oriental and black individuals. Our results indicate the importance of ethnic origin in terms of the expected HLA-A*02 allelic profile, and emphasize the functional significance of allele specific subtyping of HLA-A*02.

BAROUCH D, KRAUSA P, BODMER J, BROWNING M, MCMICHAEL A. 1995. IDENTIFICATION OF A NOVEL HLA-A2 SUBTYPE, HLA-A-ASTERISK-0216 IMMUNOGENETICS, 41 (6), pp. 388-388. | Read more

Matsui M, Moots RJ, Warburton RJ, Peace-Brewer AL, Tussey LG, Quinn DG, McMichael AJ, Frelinger JA. 1995. Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition. J Immunol, 154 (3), pp. 1088-1096. | Show Abstract

During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide.

BOWNESS P, MCMICHAEL A. 1995. IDENTIFICATION OF T-CELL RECEPTOR RECOGNITION RESIDUES FOR AN INFLUENZA-VIRUS NONAMER PEPTIDE EPITOPE PRESENTED BY HLA B27 JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 294-294.

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.

KRAUSA P, BAROUCH D, BODMER J, HILL A, MASON C, MCMICHAEL A, BROWNING M. 1995. CHARACTERIZATION OF A NOVEL HLA-A2 VARIANT, A(ASTERISK)0214, BY ARMS-PCR AND DNA-SEQUENCING IMMUNOGENETICS, 41 (1), pp. 50-50. | Read more

Krausa P, Barouch D, Bodmer JG, Hill AV, Mason C, McMichael AJ, Browning MJ. 1995. Characterization of a novel HLA-A2 variant, A*0214, by ARMS-PCR and DNA sequencing. Immunogenetics, 41 (1), pp. 50.

Barouch D, Krausa P, Bodmer J, Browning MJ, McMichael AJ. 1995. Identification of a novel HLA-A2 subtype, HLA-A*0216. Immunogenetics, 41 (6), pp. 388.

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Tussey LG, Rowland-Jones S, Zheng TS, Androlewicz MJ, Cresswell P, Frelinger JA, McMichael AJ. 1995. Different MHC class I alleles compete for presentation of overlapping viral epitopes Immunity, 3 (1), pp. 65-77. | Show Abstract | Read more

We previously identified an HLA-B8+ donor, NW, whose lymphoblestoid cells failed to present a B8-restricted epitope from the influenza A nucleoprotein following viral infection, although added peptide could still be presented. The failure to present through HLA-B8 following viral infection appears to be specific for the NP epitope. Here, we report that donor NW makes an HLA-132702-restricted influenza-specific CTL response to an epitops in the nucleoprotein that overlaps the 138-restricted epitope by 8 aa. Two mechanisms for the failure of this cell line to present the 138-restricted epitope following viral infection are investigated. One is that there is an antigen processing polymorphism specific to the NW cell line, so that there is either preferential generation or preferential transport of the B27702 epitope. The other is that 138 and 132702 compete for a common peptide fragment in the ER and this leads to suboptimal loading of HLA-138. © 1995.

MOSS P, ROWLANDJONES S, FRODSHAM P, GIANGRANDE P, MCMICHAEL A, BELL J. 1994. VERY HIGH-FREQUENCY OF HIV-SPECIFIC CYTOTOXIC T-CELLS IN HEMOPHILIA PATIENTS BLOOD, 84 (10), pp. A481-A481.

Bowness P, Allen RL, McMichael AJ. 1994. Identification of T cell receptor recognition residues for a viral peptide presented by HLA B27. Eur J Immunol, 24 (10), pp. 2357-2363. | Show Abstract | Read more

The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383-391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR V alpha 14 gene segment (but not those using TcR V alpha 12) were also sensitive to P1 substitutions, suggesting that the TcR alpha chain of these clones lies over the N terminus of bound peptide, and that the "footprint" of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are "flag" residues with TcR-accessible side chains.

Matsui M, Moots RJ, McMichael AJ, Frelinger JA. 1994. Significance of the six peptide-binding pockets of HLA-A2.1 in influenza A matrix peptide-specific cytotoxic T-lymphocyte reactivity. Hum Immunol, 41 (2), pp. 160-166. | Show Abstract | Read more

To evaluate the roles of the six peptide-binding pockets of HLA-A2.1 in FMP-specific CTL recognition, we have constructed an extensive library of HMy2.C1R cell lines expressing mutant HLA-A2.1 molecules with different amino acid substitutions in each of the six pockets. These cell lines were tested for their ability to present synthetic FMP 58-66 to FMP-specific, HLA-A2.1-restricted human CTL lines. Six of 12 mutants with amino acid changes in pocket B significantly affect the FMP-specific CTL recognition, suggesting that pocket B plays a critical role in FMP-specific CTL recognition. Surprisingly, mutations in all other pockets, except for pocket F, also have significant effects on the CTL recognition. These results suggest that even the shallow pockets, which are likely to be less critical for peptide binding than the deep pockets, play a crucial role in FMP-specific CTL recognition.

Haurum JS, Arsequell G, Lellouch AC, Wong SY, Dwek RA, McMichael AJ, Elliott T. 1994. Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes. J Exp Med, 180 (2), pp. 739-744. | Show Abstract | Read more

Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide.

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.

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COLBERT R, ROWLANDJONES S, MCMICHAEL A, FRELINGER J. 1994. DIFFERENCES IN PEPTIDE PRESENTATION BETWEEN B27 SUBTYPES - THE IMPORTANCE OF THE P1 SIDE-CHAIN IN MAINTAINING HIGH-AFFINITY PEPTIDE BINDING TO B-ASTERISK-2703 IMMUNITY, 1 (2), pp. 121-130. | Read more

Colbert RA, Rowland-Jones SL, McMichael AJ, Frelinger JA. 1994. Differences in peptide presentation between B27 subtypes: the importance of the P1 side chain in maintaining high affinity peptide binding to B*2703. Immunity, 1 (2), pp. 121-130. | Show Abstract | Read more

Susceptibility to spondyloarthropathies is strongly associated with the MHC class I molecule HLA-B27, and is hypothesized to result from the presentation of arthritogenic peptides. Subtypes of B27 that differ structurally but are disease-associated ought to be capable of presenting such peptides, while nondisease-associated subtypes would not. We demonstrate that B*2703, the predominant West African B27 subtype that may not predispose to disease, is not recognized by most B*2705-alloreactive CTL, and does not efficiently present a known B*2705-restricted influenza A nucleoprotein (NP) peptide. We show inefficient presentation is due to a reduced binding affinity of B*2703 for the NP peptide. Furthermore, substituting Arg for the naturally occurring Ser at P1 of the NP peptide, restores high affinity binding and efficient presentation by B*2703. Our results suggest that B*2703 will bind and present efficiently only a subset of the peptides that bind to B*2705, in particular those with Arg or Lys at P1. The apparent lack of disease in individuals with B*2703 may be due to an inability to bind and present putative arthritogenic peptides.

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WARBURTON R, MATSUI M, ROWLANDJONES S, GAMMON M, KATZENSTEIN G, WEI T, EDIDIN M, ZWEERINK H, MCMICHAEL A, FRELINGER J. 1994. MUTATION OF THE ALPHA(2) DOMAIN DISULFIDE BRIDGE OF THE CLASS-I MOLECULE HLA-A-ASTERISK-0201 - EFFECT ON MATURATION AND PEPTIDE PRESENTATION HUMAN IMMUNOLOGY, 39 (4), pp. 261-271. | Read more

Warburton RJ, Matsui M, Rowland-Jones SL, Gammon MC, Katzenstein GE, Wei T, Edidin M, Zweerink HJ, McMichael AJ, Frelinger JA. 1994. Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*0201. Effect on maturation and peptide presentation. Hum Immunol, 39 (4), pp. 261-271. | Show Abstract | Read more

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.

Hill AB, Barnett BC, McMichael AJ, McGeoch DJ. 1994. HLA class I molecules are not transported to the cell surface in cells infected with herpes simplex virus types 1 and 2. J Immunol, 152 (6), pp. 2736-2741. | Show Abstract

To assess the effect of herpes simplex virus (HSV) on assembly and transport of class I MHC molecules, we compared class I MHC immunoprecipitated from metabolically labeled infected and uninfected human dermal fibroblasts. The immunoprecipitates were analyzed by isoelectric focusing, allowing identification of individual class I alleles and assessment of transport through the Golgi apparatus by the sialation of carbohydrate residues. In cells infected with wild-type HSV, class I synthesis was reduced or abolished because of the host protein synthesis shutoff function of the UL41 gene product. In cells infected with mutant viruses of both HSV-2 strain G and HSV-1 strain 17 that lack the UL41 gene, class I HLA molecules failed to become sialated, suggesting that they were not transported to the Golgi apparatus. In contrast, transferrin receptor was normally sialated in both infected and uninfected cells. Drug treatments of cells to restrict viral gene expression suggested that an early gene or genes were responsible for the effect. A pulse chase showed that class I molecules were synthesized in normal amounts in infected cells, but that heavy chains were retained in a sialyl transferase negative compartment either stably associated with beta 2m or as free heavy chain in a pattern that is characteristic for each class I allele. HSV is thus the fourth example of a DNA virus that interferes with class I assembly or transport.

MURRAY N, SIMMONS D, MCMICHAEL A, BODMER W. 1994. DEVELOPMENT OF A SYSTEM OF EXPRESSION CLONING FOR MOLECULES COSTIMULATORY FOR T-CELLS JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 432-432.

Martin SJ, Vyakarnam A, Cheingsong-Popov R, Callow D, Jones KL, Senior JM, Adams SE, Kingsman AJ, Matear P, Gotch FM. 1993. Immunization of human HIV-seronegative volunteers with recombinant p17/p24:Ty virus-like particles elicits HIV-1 p24-specific cellular and humoral immune responses. AIDS, 7 (10), pp. 1315-1323. | Show Abstract | Read more

OBJECTIVE: To evaluate the immune response to HIV-1 p24 generated in vivo by p17/p24:Ty virus-like particles (p17/p24:Ty-VLP) by examining the lymphoproliferative and antibody (Ab) responses to HIV-1 p24, as well as Gag-specific cytotoxic T lymphocytes (CTL), in HIV-seronegative volunteers immunized with hybrid p17/p24:Ty-VLP. DESIGN AND METHODS: Sixteen HIV-seronegative volunteers were immunized with p17/p24:Ty-VLP at two dose levels (100 or 500 micrograms) and monitored for the following 48 weeks for production of anti-p24 and anti-p17 Ab, in vitro lymphoproliferative responses to HIV-1 p24 and p17, and in vitro CTL responses to HIV-1 Gag. RESULTS: Twelve out of the 16 volunteers had significant p24-specific proliferative responses, with volunteers on the higher dose schedule exhibiting earlier proliferative responses than those on the lower dose schedule. Proliferative responses in both volunteer groups were similar in overall magnitude but appeared at different times during the immunization schedule. Anti-p24 Ab were detected in six out of the nine individuals in the lower dose group and in five out of the seven in the higher dose group. There was a good correlation between the presence of p24-specific Ab and the detection of lymphoproliferative responses to the p24 protein in peripheral blood mononuclear cells isolated from the same individuals. Anti-p17 Ab were detected in five volunteers. No Gag-specific CTL responses were detected. CONCLUSION: We conclude that hybrid HIV-1 p17/p24:Ty-VLP are capable of inducing both cellular and humoral immunity to HIV-1 Gag p17 and p24 components and are worthy of further study as a potential HIV immunotherapeutic.

Gotch F, McAdam SN, Allsopp CE, Gallimore A, Elvin J, Kieny MP, Hill AV, McMichael AJ, Whittle HC. 1993. Cytotoxic T cells in HIV2 seropositive Gambians. Identification of a virus-specific MHC-restricted peptide epitope. J Immunol, 151 (6), pp. 3361-3369. | Show Abstract

A preliminary study of gag-specific, MHC-restricted CD8+ CTL has been performed in nine Gambian patients infected with HIV2. Such CTL were present in at least 55% of patients in fresh peripheral blood mononuclear cells without the requirement for in vitro restimulation. We have identified a nonamer peptide from HIV2 gag that is recognized by CD8+ HLA-B53 CTL using an amino acid sequence motif predicted from analysis of endogenous peptides eluted from HLA-B53 molecules. This peptide, from an HIV2/SIV conserved sequence, has previously been reported to be recognized by CTL from non-human primates vaccinated with recombinant vaccinia virus expressing the gag protein of SIV or infected with SIV virus. HLA-B53-restricted, HIV2 gag-specific CTL did not recognize target cells expressing HIV1 gag proteins, indicating that no cellular cross protection to HIV1 could be expected in this case.

MOOTS R, YOUNG S, STROMINGER J, FRELINGER J, BACON P, MCMICHAEL A. 1993. INTERACTIONS BETWEEN PEPTIDE AND CLASS-I MHC - IMPLICATIONS FOR IMMUNOTHERAPY IN RHEUMATIC DISEASE ARTHRITIS AND RHEUMATISM, 36 (9), pp. S148-S148.

Rowland-Jones SL, Powis SH, Sutton J, Mockridge I, Gotch FM, Murray N, Hill AB, Rosenberg WM, Trowsdale J, McMichael AJ. 1993. An antigen processing polymorphism revealed by HLA-B8-restricted cytotoxic T lymphocytes which does not correlate with TAP gene polymorphism. Eur J Immunol, 23 (8), pp. 1999-2004. | Show Abstract | Read more

In previous studies of antigen presentation through HLA-B27, we identified a healthy person whose lymphoblastoid cells do not present three B27-restricted viral epitopes to specific cytotoxic T lymphocytes (CTL), despite adequate cell surface expression of HLA-B2702 of normal sequence. Similar findings were observed in all members of his family sharing the HLA-A3-B2702 haplotype. The original donor, NW, carries HLA-B8 on his other class I haplotype, which his daughter, HW, has inherited. We now report a failure to present an HLA-B8-restricted epitope from influenza nucleoprotein following viral infection of NW cells, although exogenous added peptide is still presented normally. However, cells from HW, which do not carry the A3-B2702 haplotype, present the expected epitope after viral infection. Another B8-restricted epitope, from human immunodeficiency virus-gag, is presented equally well by both cell lines when infected with gag-vaccinia. This antigen processing phenotype does not correlate with any of the known human TAP-1 and TAP-2 polymorphisms.

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COLBERT R, ROWLANDJONES S, MCMICHAEL A, FRELINGER J. 1993. ALLELE-SPECIFIC B-POCKET TRANSPLANT IN CLASS-I MAJOR HISTOCOMPATIBILITY COMPLEX PROTEIN-CHANGES REQUIREMENT FOR ANCHOR RESIDUE AT P2 OF PEPTIDE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 90 (14), pp. 6879-6883. | Read more

Colbert RA, Rowland-Jones SL, McMichael AJ, Frelinger JA. 1993. Allele-specific B pocket transplant in class I major histocompatibility complex protein changes requirement for anchor residue at P2 of peptide. Proc Natl Acad Sci U S A, 90 (14), pp. 6879-6883. | Show Abstract | Read more

To investigate the role of an anchoring pocket in allele-specific peptide presentation by a major histocompatibility complex class I molecule, we "transplanted" a B pocket from HLA-A*0201 into HLA-B*2705 by site-directed mutagenesis. The resulting protein, designated B27.A2B, binds a different set of endogenous peptides than B*2705 as evidenced by complete loss of allorecognition as well as restored expression in the antigen processing-defective mutant cell line T2. B27.A2B also fails to present an HLA-B27-restricted influenza virus peptide [nucleoprotein (383-391)] to cytotoxic T lymphocytes (CTLs). However, substitution of leucine, the predominant P2 anchor residue in A*0201-restricted peptides, for arginine, the P2 anchor in nucleoprotein-(383-391) and other B*2705-restricted peptides, restores recognition of B27.A2B by the same B*2705-restricted peptide-specific CTLs. These results demonstrate that a dominant polymorphic pocket in a class I molecule, through interaction with the anchor residue of an antigenic peptide, can distinguish among peptides differing by only a single amino acid and thus determine the allelic specificity of peptide presentation.

Bowness P, Moss PA, Rowland-Jones S, Bell JI, McMichael AJ. 1993. Conservation of T cell receptor usage by HLA B27-restricted influenza-specific cytotoxic T lymphocytes suggests a general pattern for antigen-specific major histocompatibility complex class I-restricted responses. Eur J Immunol, 23 (7), pp. 1417-1421. | Show Abstract | Read more

Eight HLA B27-restricted influenza A virus nucleoprotein 383-391-specific cytotoxic T lymphocyte (CTL) clones were obtained from three unrelated donors following natural infection. T cell receptor (TcR) usage was studied using the "anchored" polymerase chain reaction. TcR alpha-chain usage was restricted with three predominant V alpha (V alpha 12.1, 14.1, 22) and two predominant J alpha segments. beta-chain variable-region usage was also conserved, with V beta 7 being used by five clones despite contributing less than 2% of peripheral blood lymphocyte V beta sequences of one individual studied. The TcR beta-chain junctional region was highly conserved even between CTL clones from unrelated individuals, with a negatively charged amino acid, contributed to by N-region addition, encoded at position 97 in all but two clones. This study shows that peptide-specific HLA B27-restricted CTL following influenza virus infection use very similar TcR and, when considered with previous studies, suggests a pattern of TcR conservation for major histocompatibility complex class I-restricted responses. No difference in TcR usage was detected between one healthy donor and two with HLA B27-associated arthritis.

Gao XM, Quinn CL, Bell JI, McMichael AJ. 1993. Expression and function of HLA-B27 in lipid-linked form: implications for cytotoxic T lymphocyte-induced apoptosis signal transduction. Eur J Immunol, 23 (3), pp. 653-658. | Show Abstract | Read more

Major histocompatibility complex (MHC) class I antigen-restricted cytotoxic T lymphocytes (CTL) kill their target cells not only by inducing irreversible membrane damage but also by triggering a programmed suicide cascade (apoptosis) in target cells. Recent evidence suggests that MHC class I antigens are involved in apoptosis signal transduction in T cells. Therefore, it is possible that MHC class I antigens are also responsible for CTL-induced signal transduction in target cells leading to apoptosis. To test this hypothesis, we have expressed HLA-B27 in Chinese hamster ovary (CHO) cells in a phosphatidyl inositol (PI) anchored form. The expressed Pl-anchored HLA-B27 (PI-B27), a 42-kDa molecule which can be cleaved off the cell surface by PI-specific phospholipase C, can function as an MHC restriction and antigen presentation element for specific CTL. Furthermore, PI-B27 transfectant CHO cells undergo rapid DNA fragmentation when pulsed with the appropriate peptide and treated with specific CTL, suggesting that the cytoplasmic and transmembrane domains of the heavy chain of class I MHC molecules are not required in CTL-induced apoptosis signal transduction in target cells.

COLBERT R, ROWLANDJONES S, MCMICHAEL A, FRELINGER J. 1993. B-POCKET TRANSPLANT FROM HLA-A2 INTO B27 CHANGES REQUIREMENT FOR P-2 ANCHOR RESIDUE IN PEPTIDE PRESENTATION JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 55-55.

HILL A, HILL A, ELVIN J, MCMICHAEL A. 1993. THE ALPHA-2 DOMAIN OF COMMON HLA B ALLELES DETERMINES A FAST OR SLOW ASSEMBLY PHENOTYPE JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 85-85.

MOOTS R, YOUNG S, STROMINGER J, FRELINGER J, BACON P, MCMICHAEL A. 1993. INTERACTIONS BETWEEN PEPTIDE AND CLASS-I MHC - IMPLICATIONS FOR IMMUNOTHERAPY IN RHEUMATIC DISEASE BRITISH JOURNAL OF RHEUMATOLOGY, 32 pp. 24-24.

Phillips RE, McMichael AJ. 1993. How does the HIV escape cytotoxic T cell immunity? Chem Immunol, 56 pp. 150-164.

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HILL A, ELVIN J, WILLIS A, AIDOO M, ALLSOPP C, GOTCH F, GAO X, TAKIGUCHI M et al. 1992. MOLECULAR ANALYSIS OF THE ASSOCIATION OF HLA-B53 AND RESISTANCE TO SEVERE MALARIA NATURE, 360 (6403), pp. 434-439. | Show Abstract | Read more

The protective association between the human leukocyte antigen HLA-B53 and severe malaria was investigated by sequencing of peptides eluted from this molecule followed by screening of candidate epitopes from pre-erythrocytic-stage antigens of Plasmodium falciparum in biochemical and cellular assays. Among malaria-immune Africans, HLA-B53-restricted cytotoxic T lymphocytes recognized a conserved nonamer peptide from liver-stage-specific antigen-1 (LSA-1), but no HLA-B53-restricted epitopes were identified in other antigens. These findings indicate a possible molecular basis for this HLA-disease association and support the candidacy of liver-stage-specific antigen-1 as a malaria vaccine component.

Bowness P, Moss PA, Tranter H, Bell JI, McMichael AJ. 1992. Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22. J Exp Med, 176 (3), pp. 893-896. | Show Abstract | Read more

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked.

Gao XM, Tite JP, Lipscombe M, Rowland-Jones S, Ferguson DJ, McMichael AJ. 1992. Recombinant Salmonella typhimurium strains that invade nonphagocytic cells are resistant to recognition by antigen-specific cytotoxic T lymphocytes. Infect Immun, 60 (9), pp. 3780-3789. | Show Abstract

To address the question of whether Salmonella-infected nonphagocytic cells could serve as target cells for recognition by antigen-specific, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL), four recombinant Salmonella typhimurium constructs that expressed full-length, or fragments of, influenza A virus nucleoprotein (NP) were made. The bacteria were shown to infect Chinese hamster ovary (CHO) cells. Appropriate major histocompatibility complex restriction molecules, HLA-B27 and H-2 Db, were transfected into CHO cells, which were then infected with recombinant S. typhimurium and used as targets for NP-specific CTL. The cells in which NP was expressed by intracellularly replicating bacteria were not lysed by NP-specific CTL, although they were killed when appropriate influenza A virus or peptides were used. Thus, S.typhimurium bacteria within nonphagocytic cells were resistant to CTL recognition. In contrast to these results, mice infected with recombinant S.typhimurium that expressed fragments of NP in the periplasm were primed for NP-specific CTL responses. The results indicate that CTL responses specific to Salmonella antigens can be generated, but the bacteria may be safe from the CTL attack once they have entered the nonphagocytic cells.

Moots RJ, Samberg NL, Pazmany L, Frelinger JA, McMichael AJ, Stauss HJ. 1992. A cross-species functional interaction between the murine major histocompatibility complex class I alpha 3 domain and human CD8 revealed by peptide-specific cytotoxic T lymphocytes. Eur J Immunol, 22 (6), pp. 1643-1646. | Show Abstract | Read more

The monomorphic cell surface glycoprotein CD8 acts as co-receptor in the recognition of peptide-major histocompatibility complex (MHC) class I complexes by cytotoxic lymphocytes (CTL) by binding to the monomorphic alpha 3 domain of the class I molecule. Positions 227 and 245 in the class I alpha 3 domain appear to be especially important for this interaction. Recent reports suggest there is no interspecies recognition between CD8 and MHC class I. In this study, hybrid genes from human class I HLA-A0201 and murine class I H-2Kb were transfected into human and mouse cells and tested in Cr-release assays using HLA-A0201-restricted influenza A matrix peptide-specific CTL as effectors. Transfected cells expressing chimeric genes comprising the alpha 1 and alpha 2 domains from HLA-A0201 together with the H-2Kb alpha 3 domain were lysed as effectively as wild-type HLA-A0201 and in both cases, killing was blocked by anti-CD8 antibody equally well. These results indicate that human CD8 can interact with the alpha 3 domain of murine class I to the same level as human class I.

Hill AV, Allsopp CE, Kwiatkowski D, Taylor TE, Yates SN, Anstey NM, Wirima JJ, Brewster DR, McMichael AJ, Molyneux ME. 1992. Extensive genetic diversity in the HLA class II region of Africans, with a focally predominant allele, DRB1*1304. Proc Natl Acad Sci U S A, 89 (6), pp. 2277-2281. | Show Abstract | Read more

Molecular HLA class II typing of greater than 1700 individuals from The Gambia in West Africa and Malawi in South-Central Africa revealed a striking diversity of HLA DRB-DQB haplotypes as defined by restriction fragment length polymorphism (RFLP); this diversity is twice as extensive as that found in northern Europeans. Despite this diversity, sequence and PCR/oligonucleotide analysis showed that the recently described variant DRB1*1304 is the commonest DRB1 allele in The Gambia. The sequence, geographical distribution, and RFLP association of this allele, together with homozygosity test results, suggest that DRB1*1304 may have arisen from DRB1*1102 and have reached its remarkably high frequency as a result of recent directional selection. The prevalence of this unusual allele has implications for trials of subunit vaccines in this area. The extensive and distinctive HLA class II region polymorphism in sub-Saharan Africans is consistent with evidence from other genetic loci implying an African origin of modern Homo sapiens.

Hill AV, Bennett S, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Brewster D, McMichael AJ, Greenwood BM. 1992. HLA, malaria and dominant protective associations. Parasitol Today, 8 (2), pp. 57. | Read more

Allsopp CE, Harding RM, Taylor C, Bunce M, Kwiatkowski D, Anstey N, Brewster D, McMichael AJ, Greenwood BM, Hill AV. 1992. Interethnic genetic differentiation in Africa: HLA class I antigens in The Gambia. Am J Hum Genet, 50 (2), pp. 411-421. | Show Abstract

A total of 752 individuals from The Gambia, west Africa who are representative of the major ethnic groups in the capital, Banjul, were serologically typed for HLA-A, -B, and -C antigens. Although all were typically "African" in their antigenic profiles, some marked frequency differences were found between the ethnic groups. Genetic distance comparisons with several other African populations showed that, although these west African populations clustered closely together, the positions of the various ethnic groups in The Gambia were consistent with historical and linguistic evidence of their affinities with one another and with other African populations. Despite the potential confounding effects both of selection by infectious diseases and of genetic drift caused by local differences in population structure, HLA frequencies appear to be of value in measuring inter- and intraregional population affinities in sub-Saharan Africa.

BOWNESS P, WORDSWORTH B, MOSS P, MCMICHAEL A, BELL J. 1992. T-CELL RECEPTOR USAGE OF SYNOVIAL LYMPHOCYTES FROM A PATIENT WITH REACTIVE ARTHRITIS BRITISH JOURNAL OF RHEUMATOLOGY, 31 pp. 40-40.

HILL A, KWIATKOWSKI D, MCMICHAEL A, GREENWOOD B, BENNETT S. 1992. MAINTENANCE OF MHC POLYMORPHISM - REPLY NATURE, 355 (6359), pp. 403-403. | Read more

Hughes AL, Nei M. 1992. Maintenance of MHC polymorphism. Nature, 355 (6359), pp. 402-403. | Read more

Hill AVS, Kwiatkowski D, McMichael AJ, Greenwood BM, Bennet S. 1992. Maintenance of MHC polymorphism [9] Nature, 355 (6359), pp. 403.

McMichael AJ. 1992. Role of class I molecules of the major histocompatibility complex in cytotoxic T-cell function in health and disease. Springer Semin Immunopathol, 14 (1), pp. 1-16. | Read more

Phillips RE, Rowland-Jones S, Nixon DF, Gotch FM, Edwards JP, Ogunlesi AO, Elvin JG, Rothbard JA, Bangham CR, Rizza CR. 1991. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature, 354 (6353), pp. 453-459. | Show Abstract | Read more

In a longitudinal study of HIV seropositive patients, there were fluctuations in the specificity of cytotoxic T cells for the virus. This was matched by variability in proviral gag DNA epitope sequences in the lymphocytes of these patients. Some of these viral variants are not recognized by autologous T cells. Accumulation of such mutations in T-cell antigenic targets would provide a mechanism for immune escape.

Moss PA, Moots RJ, Rosenberg WM, Rowland-Jones SJ, Bodmer HC, McMichael AJ, Bell JI. 1991. Extensive conservation of alpha and beta chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc Natl Acad Sci U S A, 88 (20), pp. 8987-8990. | Show Abstract | Read more

The major histocompatibility complex class I molecule HLA-A2.1 presents the influenza A virus matrix peptide 57-68 to cytotoxic T lymphocytes in all individuals with this common HLA type and is among the most thoroughly studied immune responses in humans. We have studied the T-cell receptor (TCR) heterogeneity of T cells specific for HLA-A2 and influenza A matrix peptide using the polymerase chain reaction. The usage of V alpha and V beta sequences seen on these T cells is remarkably conserved as are certain junctional sequences associated with alpha and beta chains. Furthermore, two unrelated HLA-A2 individuals have a similar pattern of TCR usage, implying that this is a predominant response in HLA-A2 populations. Analysis in one individual showed that the conserved TCR V alpha and V beta genes are minor members of the peripheral blood TCR repertoire. The sequences provide important information on the TCR necessary for the final structural analysis of this ternary complex.

HILL A, ALLSOPP C, KWIATKOWSKI D, MCMICHAEL A, GREENWOOD B. 1991. NUMEROUS AFRICAN IMMUNOGENETIC AND RED-CELL POLYMORPHISMS ARE ASSOCIATED WITH PROTECTION FROM SEVERE MALARIA AMERICAN JOURNAL OF HUMAN GENETICS, 49 (4), pp. 460-460.

Nixon DF, McMichael AJ. 1991. Cytotoxic T-cell recognition of HIV proteins and peptides. AIDS, 5 (9), pp. 1049-1059.

Hill AV, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Bennett S, Brewster D, McMichael AJ, Greenwood BM. 1991. Common west African HLA antigens are associated with protection from severe malaria. Nature, 352 (6336), pp. 595-600. | Show Abstract | Read more

A large case-control study of malaria in West African children shows that a human leucocyte class I antigen (HLA-Bw53) and an HLA class II haplotype (DRB1*1302-DQB1*0501), common in West Africans but rare in other racial groups, are independently associated with protection from severe malaria. In this population they account for as great a reduction in disease incidence as the sickle-cell haemoglobin variant. These data support the hypothesis that the extraordinary polymorphism of major histocompatibility complex genes has evolved primarily through natural selection by infectious pathogens.

Hill AV, Allsopp CE, Kwiatkowski D, Anstey NM, Greenwood BM, McMichael AJ. 1991. HLA class I typing by PCR: HLA-B27 and an African B27 subtype. Lancet, 337 (8742), pp. 640-642. | Show Abstract | Read more

We describe a rapid method of HLA class I typing using the polymerase chain reaction and oligonucleotide hybridisation that eliminates the requirements for viable lymphocytes and allows subtypes to be defined. We have used this to demonstrate that the predominant subtype of HLA-B27 in the Gambia, West Africa, is HLA-B*2703, which is very rare or absent in other racial groups. This subtype differs from the common Caucasian HLA-B27 subtypes in its recognition by cytotoxic T cells. We propose that HLA*B-2703, unlike other HLA-B27 subtypes, may not be associated with ankylosing spondylitis, thus accounting in part for the rarity of this condition in black populations.

Gotch FM, Hovell R, Delchambre M, Silvera P, McMichael AJ. 1991. Cytotoxic T-cell response to simian immunodeficiency virus by cynomolgus macaque monkeys immunized with recombinant vaccinia virus. AIDS, 5 (3), pp. 317-320. | Show Abstract | Read more

Simian immunodeficiency virus (SIV) gag-specific major histocompatibility complex (MHC)-restricted cytotoxic T-lymphocyte (CTL) activity was elicited in four out of six cynomolgus macaques after two immunizations with SIV gag recombinant vaccinia virus (rVV). No activity could be seen in three out of three non-immunized control animals. Low levels of anti-gag antibody were also seen in the same four responding animals. Virus-specific, MHC-restricted CTL are thought to give some protection and to assist in recovery in viral infection, and the induction of such CTL following vaccination with a single viral protein should act as an encouragement to those proposing similar vaccination studies in man.

Allsopp CE, Hill AV, Kwiatkowski D, Hughes A, Bunce M, Taylor CJ, Pazmany L, Brewster D, McMichael AJ, Greenwood BM. 1991. Sequence analysis of HLA-Bw53, a common West African allele, suggests an origin by gene conversion of HLA-B35. Hum Immunol, 30 (2), pp. 105-109. | Show Abstract | Read more

In the West African population of the Gambia the class I antigen HLA-Bw53 is found at high frequency. We used the polymerase chain reaction to amplify cDNA from an individual homozygous for this allele and determined the nucleotide sequence of the polymorphic alpha 1 and alpha 2 domains. The HLA-Bw53 sequence is identical to HLA-B35 except for a short sequence at the 3' end of exon 2 (encoding the alpha 1 domain) which specifies a Bw4 rather than a Bw6 motif. This suggests an origin for HLA-Bw53 involving a gene conversion of HLA-B35 by an allele containing this Bw4 sequence. The alpha 2 domain shared by HLA-Bw53, -B35, and -Bw58 is particularly common in sub-Saharan Africans.

Moots RJ, Matsui M, Pazmany L, McMichael AJ, Frelinger JA. 1991. A cluster of mutations in HLA-A2 alpha 2 helix abolishes peptide recognition by T cells. Immunogenetics, 34 (3), pp. 141-148. | Show Abstract | Read more

In order to investigate the regions of HLA-A2 that control peptide-specific cytotoxic T lymphocyte (CTL) recognition, 37 HLA-A2 genes coding for 50 point mutations that span the alpha 2 helix were synthesized by the technique of saturation mutagenesis. Twenty-nine of these genes, which code for 41 point mutations, were transfected into C1R cells and used as targets in cytotoxicity assays, in the presence of influenza-A matrix peptide 58-68 with specific CTL as effectors. All the transfectants were recognized fully by matrix peptide-specific CTL apart from those with amino acid substitutions at positions 152, 154, 155, 156, or 161, which led to a total loss of recognition and those with mutations at residue 27 or a double mutation at 138 and 150, which were recognized in an intermediate manner. The clustering of the crucial residues that emerges may reflect direct interaction of their side-chains with peptide or the CTL receptor.

Robbins PA, McMichael AJ. 1991. Immune recognition of HLA molecules downmodulates CD8 expression on cytotoxic T lymphocytes. J Exp Med, 173 (1), pp. 221-230. | Show Abstract | Read more

An HLA-A2+ cytotoxic T lymphocyte (CTL) line restricted by HLA-A2 in recognition of an influenza B virus nucleoprotein (BNP) peptide uses the CD8 coreceptor in the recognition of this viral peptide. Incubation of these CTL with BNP peptide in the absence of antigen-presenting cells downmodulates CD8 alpha and CD8 beta expression and reduces their ability to lyse target cells without inducing self-lysis. CD8 downmodulation was dependent on peptide concentration, time of exposure, and T cell receptor specificity. Another viral peptide from the influenza A virus matrix protein interacting with HLA-A2 had no effect on CD8 expression. Upon further investigation, an anti-HLA class I monoclonal antibody (mAb), anti-HLA class II mAb, and HLA alloantisera were found to downmodulate CD8 alpha and CD8 beta expression and induce CTL nonresponsiveness without causing degranulation. When CD8 alpha and CD8 beta expression was modulated by viral peptide or anti-HLA mAbs, other cell surface molecules were unchanged. Finally, incubation of peripheral blood lymphocytes with these anti-HLA mAbs induced no change in CD8 expression on resting cells but did downmodulate it on mitogen-activated cells. These results suggest that T cell recognition of the HLA-A2-BNP peptide complex on neighboring CTL may be the mechanism for CD8 downmodulation induced by the BNP viral peptide. This mechanism may be important in clonal anergy.

Bangham CR, McMichael AJ. 1990. HIV infection. Why the long latent period? Nature, 348 (6300), pp. 388. | Read more

Nixon DF, Huet S, Rothbard J, Kieny MP, Delchambre M, Thiriart C, Rizza CR, Gotch FM, McMichael AJ. 1990. An HIV-1 and HIV-2 cross-reactive cytotoxic T-cell epitope. AIDS, 4 (9), pp. 841-845. | Show Abstract | Read more

The HLA-B27-restricted HIV gag cytotoxic T-lymphocyte (CTL) epitope, 265-279, is highly conserved amongst HIV-1 isolates, only one, HIV-1ELI, having a single amino acid substitution. Over the same region HIV-2 differs by five amino acids. As a broadly cross-protective AIDS vaccine should protect against infection from all isolates of HIV-1 and HIV-2, we tested CTL specific for the HIV-1 265-279 epitope with peptide analogues from HIV-1ELI, HIV-2 and two simian immunodeficiency virus (SIV) isolates, and with recombinant vaccinia viruses expressing the respective gag genes, to determine whether there was any cross-reactivity for this CTL epitope. CTL from the HIV-1-infected donor could recognize the HIV-1ELI peptide, the HIV-2 peptide and recombinant vaccinia virus-infected target and one of the two SIV peptide-treated targets. Epitopes that exhibit such cross-reactivity may be valuable in vaccine design.

Frelinger JA, Gotch FM, Zweerink H, Wain E, McMichael AJ. 1990. Evidence of widespread binding of HLA class I molecules to peptides. J Exp Med, 172 (3), pp. 827-834. | Show Abstract | Read more

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

Ellis SA, McMichael AJ. 1990. Immunology. Tamarin compatibility. Nature, 346 (6279), pp. 17. | Read more

Bangham CR, McMichael AJ. 1990. Virology. Nosing ahead in the cold war. Nature, 344 (6261), pp. 16. | Read more

Ellis SA, Palmer MS, McMichael AJ. 1990. Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA Class I molecule. J Immunol, 144 (2), pp. 731-735. | Show Abstract

Extravillous trophoblast from normal human placenta has been shown previously to express an unusual form of HLA class I molecule, as does a choriocarcinoma cell line, BeWo. This molecule has a H chain of approximately 40 kDa and appears to be nonpolymorphic. We have isolated and sequenced a HLA class I cDNA clone, which probably corresponds to this molecule, from a library derived from BeWo. The nucleotide sequence shows a high degree of homology with the published sequence of a genomic clone, HLA 6.0, which is the product of a class I locus other than A, B, or C, (provisionally designated "HLA G"). The expressed product of this locus has not previously been localized. We have used the polymerase chain reaction to demonstrate the presence of similar HLA class I sequences in cDNA from normal extravillous trophoblast. Although there is some nucleotide sequence polymorphism the amino acid sequence of this molecule is conserved. It is therefore unlikely to provoke immune responses even though it is found at the fetal-maternal interface.

Gotch FM, Nixon DF, Alp N, McMichael AJ, Borysiewicz LK. 1990. High frequency of memory and effector gag specific cytotoxic T lymphocytes in HIV seropositive individuals. Int Immunol, 2 (8), pp. 707-712. | Show Abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) specific cytotoxic T lymphocytes (CTL) have been detected in most patients following infection. It has also been suggested that the specific CTL response, which may play an important role in delaying disease in infected humans, may decline during the course of disease progression. We have measured HIV peptide specific CTL precursor frequency by limiting dilution analysis in two healthy seropositive patients whose fresh peripheral blood mononuclear cells (PBM) specifically lysed MHC matched target cells (restricted by HLA B27 and B8 respectively). The frequency of HIV peptide specific CTL precursors is high (3 x 10(-3)-10(-4], but lower than estimates of circulating CTL with the same specific cytotoxic activity present directly in peripheral blood using the same sample (10(-2)-10(-3]. We suggest that this difference may result from terminal effector CTL differentiation in the T cell lineage. This implies that only a subset of CTL effectors have growth potential, whereas relatively higher levels of CTL with lytic activity can be detected in PBM of healthy HIV seropositive patients.

Bangham CRM, McMichael AJ. 1990. Nosing ahead in the cold war Nature, 344 (6261), pp. 16-16. | Read more

Huet S, Nixon DF, Rothbard JB, Townsend A, Ellis SA, McMichael AJ. 1990. Structural homologies between two HLA B27-restricted peptides suggest residues important for interaction with HLA B27. Int Immunol, 2 (4), pp. 311-316. | Show Abstract | Read more

Recently we described an HLA B27-restricted peptide derived from HIV gag p24 protein. In this study we have isolated an HLA B27-restricted peptide from the nucleoprotein (NP) of influenza A virus. The shortest fragment recognized by cytotoxic T lymphocyte (CTL) is eight amino acids long, residues 384-391. Comparison of the sequence of these two HLA B27 restricted peptides reveals homologies which can be aligned from one peptide to the other. Of the eight residues, two are identical: tryptophan and isoleucine. Both peptides have a positively charged residue at the N terminus, lysine at position 265 of gag and arginine at position 384 of NP. Using modified peptides we have shown that lysine or arginine is crucial for the interaction with HLA B27. The wild-type gag peptide blocked CTL recognition of NP peptide by influenza-specific CTL, but removal of the lysine prevented inhibition of NP peptide recognition. The importance of these charged residues was confirmed by the observation that truncated NP and gag peptides where the lysine or arginine was removed were not recognized by specific CTL. Further studies showed that the tryptophan residue influenced the association of the gag peptide with HLA B27, because the affinity of the gag peptide for B27 was strongly increased after replacing this residue with a leucine or a tyrosine. However, these peptides were not recognized by gag-specific CTL, suggesting that the tryptophan may interact with both HLA B27 and T cell receptor. These observations should help in the identification of HLA B27-restricted peptides from other viruses or organisms.

Ellis SA, McMichael AJ. 1990. Tamarin compatibility Nature, 346 (6279), pp. 17-17. | Read more

Bangham CRM, McMichael AJ. 1990. Why the long latent period? Nature, 348 (6300), pp. 388-388. | Read more

Robbins PA, Lettice LA, Rota P, Santos-Aguado J, Rothbard J, McMichael AJ, Strominger JL. 1989. Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2. J Immunol, 143 (12), pp. 4098-4103. | Show Abstract

An influenza B virus nucleoprotein (BNP) peptide, residues 82-94, defined by limited sequence homology with an HLA-A2-restricted peptide from influenza A matrix protein, was recognized by HLA-A2-restricted CTL. Reciprocal inhibition of T cell recognition by the two peptides suggest that the BNP peptide may have lower avidity for HLA-A2 molecules than the matrix peptide. The interaction between this peptide and HLA-A2 was explored by studying the CTL recognition of BNP 82-94 presented by mutant HLA-A2 molecules. Mutations at residues 9, 99, 70, 74, 152 and 156 were found to abolish T cell recognition of the BNP peptide. These results were compared with results previously obtained with the influenza A matrix peptide and suggest that the two peptides bind differently in the peptide binding site.

Isaacs D, Taylor CJ, Ting A, McMichael AJ. 1989. HLA class I antigens in severe RSV bronchiolitis. Tissue Antigens, 34 (3), pp. 210-212. | Show Abstract | Read more

Cytotoxic T-lymphocytes (CTL) recognize virus-infected cells in association with HLA class I antigens. There is strong evidence of the importance of CTL in respiratory syncytial virus (RSV) infections. We looked for but were unable to demonstrate an association between particular HLA class I antigens and severe RSV bronchiolitis in infants.

Reay PA, Jones IM, Gotch FM, McMichael AJ, Brownlee GG. 1989. Recognition of the PB1, neuraminidase, and matrix proteins of influenza virus A/NT/60/68 by cytotoxic T lymphocytes. Virology, 170 (2), pp. 477-485. | Show Abstract | Read more

We have investigated the recognition of the PB1, neuraminidase, and matrix (M1) proteins of influenza virus A/NT/60/68 (H3N2 subtype) by secondary in vitro stimulated polyclonal cytotoxic T lymphocyte (CTL) populations. While these three proteins have different functions and cellular locations, they can all be recognized as target antigens. However, the immunogenicity of these proteins for CTLs is under strict genetic control. Thus, PB1 protein is recognized as a cross-reactive target antigen by CTLs raised in CBA (H-2k) but not BALB/c (H-2d) mice. CBA, but not BALB/c mice, also generate a low-level CTL response to the neuraminidase. This latter response was only detectable following in vivo priming of CBA mice with a recombinant vaccinia virus expressing neuraminidase (N2-VACC). The matrix protein, expressed from recombinant vaccinia virus M-VACC, was not recognized as an antigen by CTL generated from either CBA or BALB/c strains of mice. By contrast, human HLA-A2-restricted influenza virus-specific CTLs were shown to recognize this matrix protein as a target antigen. Endogenous expression of as little as 90 amino acids of the matrix protein was sufficient to render target cells susceptible to lysis by such CTLs.

Ellis SA, Strachan T, Palmer MS, McMichael AJ. 1989. Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo. J Immunol, 142 (9), pp. 3281-3285. | Show Abstract

We have used Northern blot analysis to detect mRNA from class I HLA genes in the human choriocarcinoma cell line BeWo, which has been previously shown to express an atypical HLA class I molecule, in the absence of HLA A and B. Hybridization was seen with three class I DNA probes, the strongest being seen with the probe pC800, which corresponds to an 800-bp section of the Cw3 gene. We have made cDNA libraries from BeWo cells and screened for positive clones by using class I DNA probes. Of the clones isolated, we determined the complete sequence of one and partial sequence of five shorter clones. They all code for an identical C locus-related product, which does not correspond to published C locus sequences.

Bodmer HC, Gotch FM, McMichael AJ. 1989. Class I cross-restricted T cells reveal low responder allele due to processing of viral antigen. Nature, 337 (6208), pp. 653-655. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTL) recognize protein antigens which have been processed by the target cell and then presented in association with the relevant class I molecule of the major histocompatibility complex (MHC). Short synthetic peptides, which are able to associate directly with target cells, may substitute for these processed fragments in stimulating antigen-specific CTL responses. Using this approach, a dominant HLA-A2-restricted epitope has previously been mapped to residues 58-68 of influenza A virus matrix protein. Here we report HLA-A2-restricted CTL which are also able to recognize this short synthetic peptide in association with HLA-Aw69, but which fail to recognize HLA-Aw69 expressing cells infected with influenza A virus. Furthermore, individuals possessing HLA-Aw69 who respond to influenza A virus, do not respond to M58-68. These results imply that the low response to this epitope on infection of HLA-Aw69 individuals with influenza A is due to failure of the naturally processed product of matrix protein to associate with Aw69.

KOUP R, SULLIVAN J, LANGLADEDEMOYEN P, HOFFENBACH A, AUTRAN B, NIXON D, MCMICHAEL A, RIVIERE Y, WALKER C, MATZINGER P, LANZAVECCHIA A. 1989. 24TH FORUM IN IMMUNOLOGY - HIV-SPECIFIC CYTO-TOXIC LYMPHOCYTES-T - DISCUSSION RESEARCH IN IMMUNOLOGY, 140 (1), pp. 118-124.

Nixon DF, McMichael AJ. 1989. HIV-gag-specific cytotoxic T lymphocytes. Res Immunol, 140 (1), pp. 107-110. | Read more

Strominger JL, Santos-Aguado J, Burakoff SJ, Vega MA, Gotch FM, Robbins PA, McMichael AJ. 1989. Mutants of HLA-A2 in the analysis of its structure and function. Cold Spring Harb Symp Quant Biol, 54 Pt 1 pp. 361-363. | Read more

McMichael AJ, Gotch FM. 1989. Recognition of influenza A virus by human cytotoxic T lymphocytes. Adv Exp Med Biol, 257 pp. 109-114.

Mills KH, Nixon DF, McMichael AJ. 1989. T-cell strategies in AIDS vaccines: MHC-restricted T-cell responses to HIV proteins. AIDS, 3 Suppl 1 (Supplement), pp. S101-S110. | Read more

Pease CT, Ellis SA, McMichael AJ, Brewerton DA. 1988. Ankylosing spondylitis without B27: no evidence for gene conversion. Ann Rheum Dis, 47 (12), pp. 1001-1003. | Show Abstract | Read more

Isoelectric focusing gel electrophoresis was used to look for variant HLA molecules in five patients with HLA-B27 negative ankylosing spondylitis (AS). The isoelectric points of the HLA-A and B antigens from these patients and HLA paired controls were identical. This implies that the HLA-A and B antigens from the patients with AS and the controls are similar. Gene conversion of a nucleotide sequence from a B27 positive gene is thus unlikely to be the explanation for the existence of AS in patients who are HLA-B27 negative by alloantisera typing.

McMichael AJ, Gotch FM, Santos-Aguado J, Strominger JL. 1988. Effect of mutations and variations of HLA-A2 on recognition of a virus peptide epitope by cytotoxic T lymphocytes. Proc Natl Acad Sci U S A, 85 (23), pp. 9194-9198. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTL) specific for influenza A virus were prepared from 15 donors. Those with HLA-A2 recognized autologous or HLA-A2-matched B-lymphoblastoid cells in the presence of synthetic peptide representing residues 55-73 or 56-68 of the virus matrix protein sequence. Influenza A virus-specific CTL from donors without HLA-A2 or with an HLA-A2 variant type failed to respond to this peptide. CTL lines specific for HLA-A2 plus peptide did not lyse peptide-treated target cells from HLA-A2 variant donors. They also failed to lyse peptide-treated cells with point mutations that had been inserted into HLA-A2 at positions 62-63, 66, 152, and 156 and, in some instances, mutations at positions 9 and 70. CTL lysed peptide-treated target cells with mutations in HLA-A2 at positions 43, 74, and 107. The results imply that this defined peptide epitope therefore interacts with HLA-A2 in the binding groove so that the long alpha-helices of HLA-A2 make important contact with the peptide at positions 66, 152, and 156. Different amino acids at position 9, which is in the floor of the peptide binding groove of HLA-A2 and the closely related position 70, modulate the peptide interaction so that some T-cell clones react and some do not.

Nixon DF, Townsend AR, Elvin JG, Rizza CR, Gallwey J, McMichael AJ. 1988. HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides. Nature, 336 (6198), pp. 484-487. | Show Abstract | Read more

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.

ROBBINS P, MCMICHAEL A. 1988. VIRAL PEPTIDES THAT INTERACT WITH HLA-A2 INHIBIT CYTO-TOXIC T-CELL ACTIVITY HUMAN IMMUNOLOGY, 23 (2), pp. 139-139. | Read more

Pukrittayakamee S, Warrell DA, Desakorn V, McMichael AJ, White NJ, Bunnag D. 1988. The hyaluronidase activities of some Southeast Asian snake venoms. Toxicon, 26 (7), pp. 629-637. | Show Abstract | Read more

The hyaluronidase activities of venoms of snakes indigenous to Southeast Asia were investigated. With the exception of the venom of the Malayan krait Bungarus candidus, the elapid venoms had either little or no hyaluronidase activities, whereas the viperid venoms possessed considerable activity. A component of Russell's viper venom with hyaluronidase activities had a mol. wt of approximately 14,000. Neither MP4, a monoclonal antibody raised against the purified Russell's viper venom hyaluronidase toxin, nor a monospecific polyclonal antivenom neutralized the hyaluronidase activities of this purified hyaluronidase component of crude Russell's viper venom. The Russell's viper venom hyaluronidase activities was labile on heating and storage. The significance of these observations to envenomation and antivenom production is discussed.

Isaacs D, Bangham CR, McMichael AJ. 1987. Cell-mediated cytotoxic response to respiratory syncytial virus in infants with bronchiolitis. Lancet, 2 (8562), pp. 769-771. | Show Abstract | Read more

A cell-mediated cytotoxic response to respiratory syncytial (RSV) was demonstrated in the peripheral blood of 4 of 22 infants with acute bronchiolitis. These 4 infants were aged 3 weeks to 3 months. No such response was found in infants older than 4 months. All 4 infants with a positive response had mild infections.

Sacks SH, Bushell A, Rust NA, Karagiannis JA, Jewell DP, Ledingham JG, Wood KJ, McMichael AJ. 1987. Functional and biochemical subtypes of the haplotype HLA-DR3 in patients with celiac disease or idiopathic membranous nephropathy. Hum Immunol, 20 (2), pp. 175-187. | Show Abstract | Read more

HLA class II beta-chain polymorphism was investigated in the haplotype HLA-DR3 to determine if patients with HLA-DR3-associated diseases express normal or variant class II polymorphisms. Analysis was carried out by two-dimensional gel electrophoresis of immunoprecipitated HLA class II molecules, DNA hybridization with DR beta and DQ beta gene probes on Taq 1, Bam H1, or Rsa 1 digests, and mixed lymphocyte culture. Two subtypes of HLA-DR3 were identified in normal homozygous DR3 individuals on the basis of polymorphism in one of two DR beta chains detected, corresponding to differences in DR beta restriction fragment patterns. These polymorphisms exhibited significant linkage disequilibrium with the A1,B8,DR3 and B18,DR3 haplotypes, respectively. In proliferative experiments, cells with the B18,DR3-associated polymorphism strongly stimulated cells from donors with the B8,DR3-related polymorphism, suggesting that a T-cell epitope recognized by B8,DR3 cells lies on the B18,DR3-associated DR beta chain. In seven HLA-DR3 homozygous patients with celiac disease and three HLA-DR3-homozygous patients with idiopathic membranous nephropathy, only the normal patterns of HLA class II molecules were displayed, the B8,DR3 type occurring in all patients and the B18,DR3 type in one patient. These data suggest that celiac disease and idiopathic membranous nephropathy are not related to disease-specific HLA-DR beta or -DQ beta gene variants within the DR3 population that are revealed by these methods.

Bastin JM, Townsend AR, McMichael AJ. 1987. Specific recognition of influenza virus polymerase protein (PB1) by a murine cytotoxic T-cell clone. Virology, 160 (1), pp. 278-280. | Show Abstract | Read more

Cytotoxic T-cell clones were raised in CBA mice that recognised both A/X31 and A/JAP/305/1957 influenza virus. Here, we describe one CTL clone that recognises target cells infected with a recombinant vaccinia virus expressing influenza PB1.

Lee BS, Rust NA, McMichael AJ, McDevitt HO. 1987. HLA-DR2 subtypes form an additional supertypic family of DR beta alleles. Proc Natl Acad Sci U S A, 84 (13), pp. 4591-4595. | Show Abstract | Read more

Homozygous lymphoblastoid cell lines representing Dw subtypes of the DR2 serotype were studied for structural polymorphism at DR beta. These subtypes included Dw2, Dw12, and non-Dw2/non-Dw12. Analysis by two-dimensional gel electrophoresis showed that two DR beta genes were expressed in each cell line studied. One of these expressed genes encoded a protein that was nonpolymorphic on two-dimensional gel electrophoresis among all subtypes. The second expressed DR beta gene was polymorphic and migrated to a position on two-dimensional gels dependent on the subtype of the cell line. cDNA sequence analysis of the DR beta genes revealed a DR beta gene identical between the Dw2 and Dw12 subtypes, which correlates with the nonpolymorphic spot on two-dimensional gels. A second gene was sequenced that exhibited variability between the Dw2 and Dw12 subtypes. This variability takes the form of clustered point mutations in the first domain of the molecules. The alleles from the DR beta genes of a non-Dw2/non-Dw12 cell line, AZH, were unusual in sequence. In contrast to other DR beta alleles, the AZH genes may have been generated by a double recombinational event between two DR beta loci from a DR2 parent. The DR2 serotype may also constitute a supertypic "family," with one DR beta gene relatively nonpolymorphic, and one that varies with Dw subtype.

Lamb JR, McMichael AJ, Rothbard JB. 1987. T-cell recognition of influenza viral antigens. Hum Immunol, 19 (2), pp. 79-89. | Read more

Pukrittayakamee S, Ratcliffe PJ, McMichael AJ, Warrell DA, Bunnag D. 1987. A competitive radioimmunoassay using a monoclonal antibody to detect the factor X activator of Russell's viper venom. Toxicon, 25 (7), pp. 721-729. | Show Abstract | Read more

A radioimmunoassay (RIA) has been developed for the detection of Russell's viper venom in body fluids. This is a competitive binding technique using a monoclonal antibody directed against the factor X activator of Russell's viper venom. The sensitivity of the test in urine was 4 ng/ml, in 0.1% bovine serum albumin-phosphate buffered saline it was 20 ng/ml and in serum it was 5 micrograms/ml. This was adequate to detect venom in the serum of four patients bitten by Russell's viper. Urine from an isolated kidney preparation perfused with Russell's viper venom contained coagulant activity and was positive using the competitive RIA. Testing of sera from other envenomated patients and pure venom from seven other species of snake indigenous to Thailand revealed RIA cross reactivity between cobra venom and Russell's viper venom. In practice, the absence of coagulant activity in cobra venom clearly distinguishes between the two. Although further development is required to elucidate the serum factors interfering with this assay, this is a promising technique, which is of potential value in the diagnosis and investigation of the pathophysiology of Russell's viper envenomation.

PEASE C, ELLIS S, MCMICHAEL A, BREWERTON D. 1987. LACK OF EVIDENCE FOR GENE CONVERSION IN B27 NEGATIVE ANKYLOSING-SPONDYLITIS BRITISH JOURNAL OF RHEUMATOLOGY, 26 pp. 28-28.

Batchelor JR, McMichael AJ. 1987. Progress in understanding HLA and disease associations. Br Med Bull, 43 (1), pp. 156-183.

Ellis SA, Sargent IL, Redman CW, McMichael AJ. 1986. Evidence for a novel HLA antigen found on human extravillous trophoblast and a choriocarcinoma cell line. Immunology, 59 (4), pp. 595-601. | Show Abstract

We describe the characterization of a novel HLA Class I molecule, which we have isolated from chorionic cytotrophoblast cell membranes, and from a trophoblast-derived choriocarcinoma cell line, BeWo; classical HLA Class I antigens are not expressed on these cells. This antigen is an electrophoretically non-polymorphic glycoprotein of approximately 40,000 molecular weight, which is found in association with beta 2 microglobulin, and which is detected by monoclonal antibodies recognizing monomorphic determinants of HLA Class I. Elucidation of the nature and origin of this molecule may provide valuable information regarding the immune barrier that exists between mother and fetus.

Bangham CR, McMichael AJ. 1986. Specific human cytotoxic T cells recognize B-cell lines persistently infected with respiratory syncytial virus. Proc Natl Acad Sci U S A, 83 (23), pp. 9183-9187. | Show Abstract | Read more

The T-lymphocyte response to respiratory syncytial (RS) virus has been invoked to explain the bronchiolitis and pneumonia caused by RS virus in human infants. However, T cells also appear to play a role in protection against RS virus infection. Although RS virus-specific human lymphocytes have been demonstrated, neither the phenotype nor the function of the lymphocytes was characterized. We describe here the induction of anti-RS virus cytotoxic T lymphocytes, in both bulk culture and restimulated cell lines, from human peripheral blood. Infection of Epstein-Barr virus-transformed human B-cell lines with RS virus in vitro readily caused a persistent infection; these cells continued to synthesize RS viral proteins and secrete infectious RS virus 4 months after infection. The persistently infected cells were used both to restimulate cytotoxic-T-cell precursors and as targets for RS virus-specific cytotoxic T cells.

McMichael AJ, Gotch FM, Rothbard J. 1986. HLA B37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes. J Exp Med, 164 (5), pp. 1397-1406. | Show Abstract | Read more

Human influenza A virus-specific, cytotoxic T cells have been shown previously to recognize the virus nucleoprotein on infected cells. CTL preparations from four HLA B37-positive donors were shown to recognize a synthetic peptide that corresponded to amino acids 335-349 of the nucleoprotein sequence. Influenza-specific CTL from 10 donors of other HLA types failed to recognize this epitope. CD8+ CTL lines were derived from lymphocytes of two HLA B37-positive donors and used to show that the peptide was represented on virus-infected cells and to determine the probable boundaries of the epitope.

Rust NA, McMichael AJ. 1986. Simplification of two-dimensional gel patterns of HLA class II antigens. Tissue Antigens, 28 (2), pp. 72-83. | Show Abstract | Read more

Two-dimensional gel electrophoresis of material immunoprecipitated from radiolabeled cells by anti-HLA class II monoclonal antibodies has been a useful technique but the patterns observed have varied considerably between laboratories. In this study we investigated the parameters that vary between different laboratories. We found that heterogeneity due to post synthetic modification of beta and alpha chains of DR and DQ can be avoided by labeling cells for 30 min with 35S-methionine. A mixture of ampholytes pH range 3.5-10:5-7 of 1:4 run for 3000 V/h gave the best resolution of beta and alpha chains. With this technique we could clearly reveal differences in the DR beta-2 chains of those DR2 haplotypes, Dw2, Dw12 and LD'tb24. The Dr beta 1 position varied between different DR types.

McMichael AJ, Michie CA, Gotch FM, Smith GL, Moss B. 1986. Recognition of influenza A virus nucleoprotein by human cytotoxic T lymphocytes. J Gen Virol, 67 ( Pt 4) (4), pp. 719-726. | Show Abstract | Read more

A recombinant vaccinia virus (NP-VAC) containing cDNA corresponding to segment 5, the nucleoprotein (NP) gene of influenza A/PR/8/34 virus was used to examine the specificity of human influenza virus immune cytotoxic T lymphocytes (CTL). Effector cell preparations from two donors recognized autologous lymphocytes that had been infected with NP-VAC. Lysis was specific because cells infected with vaccinia virus were not killed and recognition was HLA-restricted. In one donor, the influenza virus-specific CTL response changed with time so that his effector cells no longer recognized autologous lymphocytes infected with NP-VAC. However, a component that was NP-specific remained because these CTL lysed the more sensitive autologous B lymphoblastoid cells that had been infected with NP-VAC. In four other donors, no NP-specific CTL response could be detected using autologous lymphocyte targets. Thus NP, an internal virus protein, is one antigen that is recognized by human influenza A virus-specific CTL, but it is likely that other individual virus components contribute to the total CTL response.

Townsend AR, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 1986. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell, 44 (6), pp. 959-968. | Show Abstract | Read more

A proportion of cytotoxic T lymphocytes (CTL) responding to infection by influenza recognize target cells that express the viral nucleoprotein. Recent work showed that CTL can recognize short overlapping regions of large nucleoprotein fragments expressed in transfected L cells. This led to the suggestion that CTL recognize segmental epitopes of denatured or degraded proteins in a similar way to helper T cells. One corollary of this idea is that CTL should recognize appropriate short peptides on the target cell surface. We demonstrate that the epitopes of nucleoprotein recognized by CTL in association with class I molecules of the major histocompatibility complex in both mouse and man can be defined with short synthetic peptides derived from the nucleoprotein sequence.

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Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 1986. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides Cell, 44 (6), pp. 959-968. | Show Abstract | Read more

A proportion of cytotoxic T lymphocytes (CTL) responding to infection by influenza recognize target cells that express the viral nucleoprotein. Recent work showed that CTL can recognize short overlapping regions of large nucleoprotein fragments expressed in transfected L cells. This led to the suggestion that CTL recognize segmental epitopes of denatured or degraded proteins in a similar way to helper T cells. One corollary of this idea is that CTL should recognize appropriate short peptides on the target cell surface. We demonstrate that the epitopes of nucleoprotein recognized by CTL in association with class I molecules of the major histocompatibility complex in both mouse and man can be defined with short synthetic peptides derived from the nucleoprotein sequence. © 1986.

Ellis SA, Taylor C, Hildreth JE, McMichael AJ. 1985. An HLA class I specific monoclonal antibody that fails to bind to all HLA-A antigens. Hum Immunol, 13 (1), pp. 13-19. | Show Abstract | Read more

A monoclonal antibody, MHM.5, specific for HLA class I antigens, bound to lymphocytes of all donors tested and was thought to bind to a monomorphic determinant. When the antibody was used to precipitate 35S methionine labeled HLA class I molecules from lymphoid cells, which were then isoelectric focused, it was found that the HLA-A1,A2 and A3 antigens were not precipitated. Similarly, MHM.5, which is IgG1, failed to block complement mediated lysis by alloantisera specific for HLA-A1, 2 and 3, and most other HLA-A antigens. HLA-Aw24, A25, and A32, and all other HLA-B and C typing reactions tested were blocked. Thus the antibody binds to an epitope that is lacking on most A antigens, but present on Aw24, A25, A32 and all B and C locus antigens. Comparison of the published amino acid sequences of HLA-A2, A3, Aw24, A28, Cw3, B7, and B40 suggests some possible sites for this epitope.

MIGUEL J, DECASTRO J, MATUTES E, RODRIGUEZ B, POLLI N, ZOLA H, MCMICHAEL A, BOLLUM F, THOMPSON D, GOLDMAN J, CATOVSKY D. 1985. CHARACTERIZATION OF BLAST CELLS IN CHRONIC GRANULOCYTIC-LEUKEMIA IN TRANSFORMATION, ACUTE MYELOFIBROSIS AND UNDIFFERENTIATED LEUKEMIA .2. STUDIES WITH MONOCLONAL-ANTIBODIES AND TERMINAL TRANSFERASE BRITISH JOURNAL OF HAEMATOLOGY, 59 (2), pp. 297-309. | Read more

Gotch FM, Kelly C, Ellis SA, Wallace L, Rickinson AB, van der Poel J, Crumpton MJ, McMichael AJ. 1985. Characterization of the HLA-A2.2 subtype: T cell evidence for further heterogeneity. Immunogenetics, 21 (1), pp. 11-23. | Show Abstract | Read more

Five blood donors were identified whose HLA-A2 is different from the common HLA-A2. Their A2 molecule (A2.2) had a more basic isoelectric point than normal A2 (A2.1). Cytotoxic T lymphocytes (CTL) restricted by HLA-A2.1, specific for influenza A and Epstein-Barr viruses, failed to lyse virus-infected target cells with HLA-A2.2. Identical patterns were obtained with both viruses. CTL from four of the A2.2-positive donors recognized target cells prepared from others in the group that shared only the HLA-A2.2 antigen. The A2.2 antigen from one donor seemed to be different in that target cells were not recognized by CTL from donors with the normal A2.1 nor with basic A2.2. There seems, therefore, to be heterogeneity within the HLA-A2.2 subtype.

Dongworth DW, Gotch FM, Hildreth JE, Morris A, McMichael AJ. 1985. Effects of monoclonal antibodies to the alpha and beta chains of the human lymphocyte function-associated (H-LFA-1) antigen on T lymphocyte functions. Eur J Immunol, 15 (9), pp. 888-892. | Show Abstract | Read more

The ability of two antibodies, one specific for the alpha chain, p180, and the other for the beta chain, p95, of the human lymphocyte function-associated (LFA-1) antigens, to inhibit T cell function was measured. Both antibodies inhibited T cell-mediated lysis of virus-infected target cells and of K562 cells. Only the anti-beta chain antibody inhibited natural killer cell lysis of K562. The antibodies inhibited cytotoxic T lymphocyte cell (CTL) lysis of HLA-mismatched target cells in the presence of concanavalin A at 6.25-12.5 micrograms/ml, but at higher doses of Con A no inhibition was seen. When the lytic process was divided into calcium-independent (adherence) and -dependent (lysis) steps the antibodies were found to block at the initial step of conjugate formation. The effects of these antibodies on T cell proliferative responses showed that responses to antigens, alloantigens, mitogens and anti-CD3 (UCHT1) antibody were greatly inhibited. All of these responses are adherent cell dependent and proliferation of adherent cell-depleted mononuclear cells to Sepharose-coupled UCHT1 was not inhibited by anti-LFA-1 antibodies. Proliferation to paired anti-CD2 (T11) antibodies was also only weakly inhibited. Release of interferon-gamma by CTL on contact with target cells was also inhibited by anti-LFA antibody. These results are evidence that the LFA antigen is necessary for a nonspecific interaction with antigen-presenting cells that is essential for activation of T cells through the CD3-T cell receptor complex.

Townsend AR, McMichael AJ. 1985. Specificity of cytotoxic T lymphocytes stimulated with influenza virus. Studies in mice and humans. Prog Allergy, 36 pp. 10-43. | Show Abstract

Doherty et al. first demonstrated that cytotoxic T lymphocytes (CTLs) specific for virus-infected cells were restricted in their recognition of target antigens by class-I antigens of the major histocompatibility complex (MHC). Since that time this phenomenon has been thoroughly explored with the result that much is now known of the self-recognising properties of CTLs and rather less is understood about the recognition of virus antigen. Until the latter has been precisely characterised, an understanding of how CTLs recognise their target is unlikely. In this review we discuss the recognition of influenza virus antigens in association with MHC class-I molecules in both mouse and man. Precise mapping of the epitopes seen by CTL receptors is now possible with important practical implications for the development of vaccins.

Mitchell DM, McMichael AJ, Lamb JR. 1985. The immunology of influenza. Br Med Bull, 41 (1), pp. 80-85.

Redman CW, McMichael AJ, Stirrat GM, Sunderland CA, Ting A. 1984. Class 1 major histocompatibility complex antigens on human extra-villous trophoblast. Immunology, 52 (3), pp. 457-468. | Show Abstract

Using immunohistological techniques, class 1 products of the major histocompatibility complex (MHC) have been demonstrated on various forms of human extra-villous trophoblast in placental tissues taken from first, second and third trimester pregnancies. This contrasts with the absence of class 1 MHC antigens on all forms of villous trophoblast. The extra-villous trophoblast which reacted with antibodies to monomorphic class 1 MHC antigens consistently failed to bind HLA-A or HLA-B antibodies specific for the foetal phenotype. This suggests, but does not prove, that the MHC antigen expression of trophoblast may be restricted to HLA-C or some other undefined class 1 antigen.

Townsend AR, McMichael AJ, Carter NP, Huddleston JA, Brownlee GG. 1984. Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell, 39 (1), pp. 13-25. | Show Abstract | Read more

L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.

Dongworth DW, McMichael AJ. 1984. Inhibition of human T lymphocyte function with monoclonal antibodies. Br Med Bull, 40 (3), pp. 254-261.

Hildreth JE, Gotch FM, Hildreth PD, McMichael AJ. 1983. A human lymphocyte-associated antigen involved in cell-mediated lympholysis. Eur J Immunol, 13 (3), pp. 202-208. | Show Abstract | Read more

Human lymphocyte function antigen (HLFA) is a cell surface protein defined by two monoclonal antibodies MHM23 and MHM24. It is present on both B and T lymphocytes but in greater amounts on the latter. Both antibodies precipitated antigen, from radiolabeled HSB-2 cells, which ran as two chains on sodium dodecyl sulfate polyacrylamide gel electrophoresis at 180 and 94 kDa. Neither antibody inhibited binding of the other, indicating that distinct epitopes were recognized. Both antibodies were shown to inhibit HLA-restricted lysis of influenza virus-infected and Epstein-Barr virus-transformed target cells by cytotoxic T lymphocytes. Blocking occurred at the level of the effector cells and in the presence of subsaturating concentrations of antibody. Both reagents also inhibited lysis of K562 cells, mediated by natural killer cells. These blocking effects differ from the inhibitory effects of monoclonal anti-HLA ABC and anti-suppressor cytotoxic T cell antibodies which inhibit only HLA-restricted lysis when present in saturating amounts. It is concluded therefore that HLFA is likely to be involved in the nonspecific adherence or lytic functions of killer cells rather than specific antigen recognition.

MIGUEL J, OBRIEN M, POLLI N, CASTRO J, ZOLA H, MCMICHAEL A, GREAVES M, GOLDMAN J, CATOVSKY D. 1983. CHARACTERIZATION OF BLAST CELLS BY ULTRASTRUCTURAL CYTO-CHEMISTRY AND MONOCLONAL-ANTIBODIES BRITISH JOURNAL OF HAEMATOLOGY, 55 (1), pp. 190-190.

McMichael AJ, Gotch FM, Noble GR, Beare PA. 1983. Cytotoxic T-cell immunity to influenza. N Engl J Med, 309 (1), pp. 13-17. | Show Abstract | Read more

In a study designed to determine whether cytotoxic T lymphocytes contribute to immunity against influenza virus infection, we inoculated 63 volunteers intranasally with live unattenuated influenza A/Munich/1/79 virus. Over the next seven days clinical observations were made, and the amount of virus shed was measured. The protective effects of preinfection serum antibody and of cytotoxic T-cell immunity against influenza A virus were assessed for each participant. All subjects with demonstrable T-cell responses cleared virus effectively. This response was observed in volunteers in all age groups, including those born after 1956, who did not have specific antibody and hence had probably not been exposed to this subtype of influenza A virus before. Cytotoxic T cells show cross-reactivity in their recognition of the different subtypes of influenza A virus, in contrast to the antibody response that is specific for each virus subtype. We conclude that cytotoxic T cells play a part in recovery from influenza virus infection.

McMichael AJ, Gotch FM, Dongworth DW, Clark A, Potter CW. 1983. Declining T-cell immunity to influenza, 1977-82. Lancet, 2 (8353), pp. 762-764. | Show Abstract | Read more

Influenza-A-virus-specific, HLA-restricted, cytotoxic T-cell immunity, measured in 189 volunteers in the six years 1977-82, showed a sharp decline from 1978. It is shown that natural infection with influenza-A virus boosts cytotoxic T-cell memory. The decline in T-cell immunity is probably associated with the low prevalence of influenza-A-virus infection since 1978.

Makgoba MW, Fuggle SV, McMichael AJ, Morris PJ. 1983. HLA-DR products are a subset of human Ia antigens. Nature, 301 (5900), pp. 531-532. | Show Abstract | Read more

Human Ia antigens are polymorphic cell-surface sialoglycoproteins which have restricted tissue distribution. They are bimolecular complexes of 34,000 (alpha) and 28,000 (beta) molecular weight and most of the polymorphism is found in the smaller polypeptides. They are involved in the initiation of immune responses and particular Ia antigens are associated with increased susceptibility to certain diseases. They are also the major barrier to human allogeneic tissue transplantation. Whereas serological analysis and mixed lymphocyte typing have defined three polymorphic families of Ia antigens, HLA-DR, -DC and -SB, protein sequencing results and studies with monoclonal antibodies indicate that the complexity is much greater. Thus the HLA-DR and DC specificities as defined by alloantisera, could represent groups of antigens which are controlled by HLA genes in linkage disequilibrium. Here, we have used a monoclonal antibody specific for HLA-DR2 to show that this determinant is carried by molecules which are distinct from those of the DC series and which represent 30% of the Ia antigens expressed on the cell surface of an HLA homozygous line PGF.

MCMICHAEL A. 1983. HYBRIDOMAS IN CANCER-DIAGNOSIS AND TREATMENT - MITCHELL,MS, OETTGEN,HF QUARTERLY REVIEW OF BIOLOGY, 58 (2), pp. 308-309. | Read more

Makgoba MW, Hildreth JE, McMichael AJ. 1983. Identification of a human Ia antigen that is different from HLA-DR and DC antigens. Immunogenetics, 17 (6), pp. 623-635. | Show Abstract | Read more

A monoclonal antibody, MHM4, identified a cell surface antigen present on B cells and not resting T cells. It precipitated two polypeptide chains of 34 000 and 28 000 daltons from B lymphoblastoid cells. This antibody bound to all B-cell lines tested, except those homozygous for HLA-DR7. Saturation binding assays and Scatchard plots of MHM4 binding to cells that did not carry HLA-DR7 indicated that this antibody bound less than the total surface Ia antigen. When the antibody was competed with eight other HLA-D-specific antibodies, the epitope recognized was shown to be distinct. Two-dimensional gel analysis revealed that a simple pattern of spots was precipitated, unlike the complex patterns obtained with other HLA-D-specific antibodies. The alpha and beta spots were different from those precipitated by HLA-DR- or DC-specific antibodies. It is argued that the MHM4 antigen is the product of an HLA locus that is distinct from HLA-DR and DC. Its relationship with HLA-SB is discussed.

Vainchenker W, Deschamps JF, Bastin JM, Guichard J, Titeux M, Breton-Gorius J, McMichael AJ. 1982. Two monoclonal antiplatelet antibodies as markers of human megakaryocyte maturation: immunofluorescent staining and platelet peroxidase detection in megakaryocyte colonies and in in vivo cells from normal and leukemic patients. Blood, 59 (3), pp. 514-521.

McMichael AJ, Gotch FM, Hildreth JE. 1982. Lysis of allogeneic human lymphocytes by nonspecifically activated T-like cells. Eur J Immunol, 12 (12), pp. 1002-1005. | Show Abstract | Read more

In the generation of cytotoxic effector cells specific for influenza A virus-infected lymphocytes, three donors have given an unusual pattern of lytic activity, killing HLA-mismatched target cells. This has been analyzed in detail for one donor and one of the other two shows similar results. Activation only requires culture in medium between 1 and 4 days and parallels development of cell line K562-directed natural killer cells. Target lymphocytes do not need to be virus-infected and appear to be normal lymphocytes. The effector cells carry the surface markers T3 and T8 defined by OKT3/anti-Leu4 and OKT8/anti-Leu2a monoclonal antibodies, respectively. Unlike HLA class 1-restricted or -directed cytotoxic T cells, neither anti-Leu2a/nor anti-Leu4 blocked killing in the absence of complement. MHM23, a monoclonal antibody specific for the human lymphocyte function antigen, blocked lysis. The results indicate that these effector cells are related to cytotoxic T lymphocytes, but can lyse allogeneic target cells through a different recognition process. There is some specificity because autologous cells were not killed.

Vainchenker W, Guichard J, Deschamps JF, Bouguet J, Titeux M, Chapman J, McMichael AJ, Breton-Gorius J. 1982. Megakaryocyte cultures in the chronic phase and in the blast crisis of chronic myeloid leukaemia: studies on the differentiation of the megakaryocyte progenitors and on the maturation of megakaryocytes in vitro. Br J Haematol, 51 (1), pp. 131-146. | Show Abstract | Read more

Megakaryocyte (MK) colony formation has been studied in the chronic phase and in the blast crisis of chronic myeloid leukaemia (CML). Blood cells were grown in plasma clot for 13 d. MKs were subsequently identified by immunofluorescent techniques using two monoclonal antiplatelet antibodies (AN51 and J15). The maturation process was studied by ultrastructural methods. A marked increase in the number of circulating CFU-MK was observed in all the 10 cases studied prior to chemotherapy (70-fold increase per ml of blood). No significant modification in the regulation of MK colony formation as compared to that of normal subjects was observed. The predominant abnormality in maturation in culture was the occurrence of many hypoploid MKs (microMKs). However, the cytoplasmic maturation of the MKs was identical to that of normal subjects with occasional platelet shedding. Since microMKs predominated in some patients, scoring of MK colonies in CML necessitated immunofluorescent labelling to permit identification of MKs. During the blast crisis, MK colony formation occurred in four out of five patients with an extremely high plating efficiency in the case of promegakaryoblastic transformation. In contrast, MK colonies could not be grown from blood samples of patients with acute leukaemia, including two cases of promegakaryoblastic leukaemia. Maturation of MKs in blast crisis was identical to that of the chronic phase. Furthermore, after short periods of culture in liquid medium, circulating promegakaryoblasts from patients in blast crisis matured with the consequent production of alpha-granules and demarcation membranes. These results confirm the contention that CML represents a pluripotent stem-cell disease, involving the MK lineage, and suggest that the block in maturation during the acute phase can be overcome in vitro.

McMichael AJ, Askonas BA, Webster RG, Laver WG. 1982. B-cell or T-cell immunity? Immunol Today, 3 (10), pp. 255-260. | Show Abstract | Read more

The influenza virus behaves unpredictably and can cause devastating pandemics. Nearly 50 years after its first isolation it is probably the most infectious agent known that we cannot yet control. Why? The answer lies in the virus's unique capacity to alter antigenically and in the inability of the host's immune system to respond satisfactorily to the vaccines currently available. What follows is a record, prepared exclusively for Immunology Today, of a conference held in Oxford on March 21-24 this year, to discuss problems relating to protection against influenza.

Wallace LE, Moss DJ, Rickinson AB, McMichael AJ, Epstein MA. 1981. Cytotoxic T cell recognition of Epstein-Barr virus-infected B cells. II. Blocking studies with monoclonal antibodies to HLA determinants. Eur J Immunol, 11 (9), pp. 694-699. | Show Abstract | Read more

Monoclonal antibodies specifically binding common determinants on all HLA-A, B anc C antigen molecules blocked the lysis of EB virus-transformed target cells by EB virus-specific cytotoxic T cells reactivated in vitro. Blocking was mediated through binding of the antibodies to the target rather than to the effector cells and was maximal (75 to 85% inhibition of lysis) at saturating antibody concentrations. A similar blocking effect was also shown by a monoclonal antibody binding to beta 2-microglobulin, a molecule physically associated with HLA-A, B and C antigens on the target cell surface, but not by a monoclonal antibody binding to the non-HLA-associated leukocyte-common antigen. Saturating concentrations of monoclonal antibodies specific for common determinants on all HLA-DRw antigen molecules either had no effect at all upon EB virus-specific T cell cytotoxicity or caused a slight, but nonspecific, inhibition. The results demonstrate unequivocally that HLA-A, B and C antigens on the target cell surface are indeed the polymorphic elements which impose genetic restriction upon EB virus-specific cytotoxic T cell function.

HILDRETH J, MCMICHAEL A. 1981. INTERACTION BETWEEN INFLUENZA-VIRUS AND HLA ANTIGENS IMMUNOBIOLOGY, 159 (1-2), pp. 36-36.

McMichael AJ, Rust NA, Pilch JR, Sochynsky R, Morton J, Mason DY, Ruan C, Tobelem G, Caen J. 1981. Monoclonal antibody to human platelet glycoprotein I. I. Immunological studies. Br J Haematol, 49 (4), pp. 501-509. | Show Abstract | Read more

A monoclonal hybridoma antibody specific for platelet glycoprotein I complex is described. The nature od the antigen was determined by demonstration that it was chymotrypsin sensitive and gave a peak at 150 000 daltons on SDS-PAGE after immunoprecipitation. The expression of the antigen is restricted to platelets and megakaryocytes with at least 1.6 x 10(4) molecules of antigen per platelet. The antibody failed to bind to platelets from patients with Bernard Soulier syndrome, where there is known to be a deficiency of glycoprotein Ib/Is expression. Binding to platelets from patients with Glanzmann's thrombasthenia was normal.

Ruan C, Tobelem G, McMichael AJ, Drouet L, Legrand Y, Degos L, Kieffer N, Lee H, Caen JP. 1981. Monoclonal antibody to human platelet glycoprotein I. II. Effects on human platelet function. Br J Haematol, 49 (4), pp. 511-519. | Show Abstract | Read more

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.

Bastin JM, Granger S, Tidman N, Janossy G, McMichael AJ. 1981. Recognition of a human T-lymphocyte differentiation antigen by an IgM monoclonal antibody. Clin Exp Immunol, 46 (3), pp. 597-606. | Show Abstract

A monoclonal antibody directed at a determinant on human T cells was produced and characterized. This IgM antibody, MBG6, bound to human peripheral blood T lymphocytes and to medullary thymocytes. It was unreactive with normal B cells, B-cell lines and granulocytes. Apart from T lymphocytes, bone marrow cells (including cells positive for the terminal transferase marker, myeloid colony-forming cells, myeloblasts, and differentiating myeloid and erythroid cells) were negative. Peripheral blood cells that were treated with MBG6 and rabbit complement were no longer capable of proliferating in response to phytohaemagglutinin or concanavalin A; MBG6 did not have any direct mitogenic action on T lymphocytes. Double immunofluorescence studies using IgM MBG6 and OKT3, and IgG2a monoclonal antibody that recognizes all peripheral T cells, showed that these two antibodies identified exactly the same cell populations. Competitive binding studies, however, indicated that MBG6 and OKT3 recognized different epitopes. The antibody may have clinical applications in bone marrow transplantation.

McMichael AJ, Gotch F, Cullen P, Askonas B, Webster RG. 1981. The human cytotoxic T cell response to influenza A vaccination. Clin Exp Immunol, 43 (2), pp. 276-284. | Show Abstract

The human cytotoxic T lymphocyte (CTL) response to challenge with influenza A vaccine was studied. Six of eight volunteers given killed whole influenza virus A/USSR (H1N1) vaccine showed substantial increases on the level of CTL memory 1 month after immunization. The CTL measured at this time showed complete cross-reactivity in their specificity for influenza A/USSR (H1N1) and A/X31 (H3N2) infected cells and also showed HLA restriction. The level of CTL memory increased in only three out of nine donors given subunit vaccine and showed no change in those not given vaccine. If cytotoxic T cells are important in influenza prophylaxis, killed whole virus vaccine should offer better protection than subunit vaccine.

McMichael AJ, Parham P, Rust N, Brodsky F. 1980. A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17. Hum Immunol, 1 (2), pp. 121-129. | Show Abstract | Read more

A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.

McMichael AJ, Bastin JM. 1980. Clinical applications of monoclonal antibodies. Immunol Today, 1 (3), pp. 56-61. | Show Abstract | Read more

Monoclonal antibodies offer many distinct advantages over conventional antisera, the most obvious being their precise specificity for a single epitope on a single antigen and their potentially unlimited supply. The quantity and purity of available antibodies facilitates antigen purification by affinity chromatography. Monoclonal antibodies can also be used to characterize different parts of a macromolecule with regard to antigenicity, functional activity or genetic variability. Here Andrew McMichael and Judy Bastin concentrate on the potential value of monoclonal antibodies in clinical medicine, surveying papers published up to June 1980, and a number of preprints and personal communications, kindly made available to them by colleagues.

McMichael AJ. 1980. HLA restriction of human cytotoxic T cells. Springer Semin Immunopathol, 3 (1), pp. 3-22. | Read more

McMichael AJ, Parham P, Brodsky FM, Pilch JR. 1980. Influenza virus-specific cytotoxic T lymphocytes recognize HLA-molecules. Blocking by monoclonal anti-HLA antibodies. J Exp Med, 152 (2 Pt 2), pp. 195s-203s. | Show Abstract

The effect of monoclonal anti-HLA antibodies on lysis of influenza virus-infected target cells by sensitized cytotoxic T lymphocytes was measured. Lysis was blocked, provided the antibody was present in a concentration sufficient to saturate the HLA determinants on the target cells. Antibodies to monomorphic HLA determinants and to beta 2-microglobulin inhibited. Antibody to HLA A2 and B17 blocked lysis of target cells that shared these antigens with the effector cell. It was concluded that the restriction elements for influenza virus-specific cytotoxic T cells are the HLA molecules themselves.

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McMichael AJ, Parham P, Brodsky FM, Pilch JR. 1980. Influenza virus-specific cytotoxic T lymphocytes recognize HLA molecules. Blocking by monoclonal anti-HLA antibodies Journal of Experimental Medicine, 152 (2 II), pp. 195-203. | Show Abstract

The effect of monoclonal anti-HLA antibodies on lysis of influenza virus-infected target cells by sensitized cytotoxic T lymphocytes was measured. Lysis was blocked, provided the antibody was present in a concentration sufficient to saturate the HLA determinants on the target cells. Antibodies to monomorphic HLA determinants and to β2-microglobulin inhibited. Antibody to HLA A2 and B17 blocked lysis of target cells that shared these antigens with the effector cell. It was concluded that the restriction elements for influenza virus-specific cytotoxic T cells are the HLA molecules themselves.

McMichael AJ, Pilch JR, Galfré G, Mason DY, Fabre JW, Milstein C. 1979. A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody. Eur J Immunol, 9 (3), pp. 205-210. | Show Abstract | Read more

Spleen cells from a BALB/c mouse that had been immunized with human thymocytes were fused with the myeloma line P3-NS 1/1 Ag 4.1. One of the resulting hybrid clones (NA 1/34) secreted an antibody that was highly specific for human thymocytes. Eighty-five % of thymocytes expressed the antigen designated HTA1. There were an estimated 15 x 10(4) molecules of HTA 1 per cell, and it is therefore a major surface molecule. The expression of this antigen on thymocytes appears to be reciprocal to HLA, as recognized by another monoclonal antibody W6/32. Immunoprecipitated material from [125I]-labeled thymocyte membranes was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate which disclosed a single component of 45,000 molecular weight.

MCMICHAEL A, WEATHERALL D. 1978. HLA COMPLEX AND THE IMMUNE-RESPONSE TO INFLUENZA-VIRUS QUARTERLY JOURNAL OF MEDICINE, 47 (188), pp. 574-574.

McMichael AJ, Askonas BA. 1978. Influenza virus-specific cytotoxic T cells in man; induction and properties of the cytotoxic cell. Eur J Immunol, 8 (10), pp. 705-711. | Show Abstract | Read more

Human peripheral blood lymphocytes have been sensitized in vitro to influenza virus antigen. After an induction period of 4--14 days, cytotoxic cells which lyse autologous influenza virus-infected lymphoid cells could be demonstrated. The cytotoxic cell is a T lymphocyte which shows specificity for sensitizing influenza virus type A or B. It cannot distinguish between major subtypes of influenza A virus. The use of virus-infected normal lymphoid cells as target cells overcame the difficulties of nonspecific killing encountered with some transformed cells.

McMichael AJ, Ting A, Zweerink HJ, Askonas BA. 1977. HLA restriction of cell-mediated lysis of influenza virus-infected human cells. Nature, 270 (5637), pp. 524-526. | Show Abstract | Read more

MURINE T lymphocytes that mediate the lysis of virus-infected cells show specificity both for the viral cell surface antigens and for the H-2K or D antigens of the major histo-compatibility complex1-8. The cytotoxic T lymphocytes and the target cell must share H-2K or D products. The experiments reported here demonstrate that there is a similar requirement for partial HLA identity between human cytotoxic lymphocytes and influenza virus-infected target cells. © 1977 Nature Publishing Group.

Simon AK, Hollander GA, McMichael A. 2015. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci, 282 (1821), pp. 20143085. | Show Abstract | Read more

This article reviews the development of the immune response through neonatal, infant and adult life, including pregnancy, ending with the decline in old age. A picture emerges of a child born with an immature, innate and adaptive immune system, which matures and acquires memory as he or she grows. It then goes into decline in old age. These changes are considered alongside the risks of different types of infection, autoimmune disease and malignancy.

Duncan CJA, Russell RA, Baxter AE, McMichael AJ, Sattentau QJ. 2012. Phagocytic uptake of transmitted/founder virus-infected CD4+T cells enhances macrophage infection HIV MEDICINE, 13 pp. 29-29.

Ritchie AJ, Cai F, Smith NM, Chen S, Song H, Brackenridge S, Abdool Karim SS, Korber BT, McMichael AJ, Gao F, Goonetilleke N. 2014. Recombination-mediated escape from primary CD8+ T cells in acute HIV-1 infection. Retrovirology, 11 (1), pp. 69. | Show Abstract | Read more

BACKGROUND: A major immune evasion mechanism of HIV-1 is the accumulation of non-synonymous mutations in and around T cell epitopes, resulting in loss of T cell recognition and virus escape. RESULTS: Here we analyze primary CD8+ T cell responses and virus escape in a HLA B*81 expressing subject who was infected with two T/F viruses from a single donor. In addition to classic escape through non-synonymous mutation/s, we also observed rapid selection of multiple recombinant viruses that conferred escape from T cells specific for two epitopes in Nef. CONCLUSIONS: Our study shows that recombination between multiple T/F viruses provide greater options for acute escape from CD8+ T cell responses than seen in cases of single T/F virus infection. This process may contribute to the rapid disease progression in patients infected by multiple T/F viruses.

Liu MK, Hawkins N, Ritchie AJ, Ganusov VV, Whale V, Brackenridge S, Li H, Pavlicek JW et al. 2013. Vertical T cell immunodominance and epitope entropy determine HIV-1 escape. J Clin Invest, 123 (1), pp. 380-393. | Show Abstract | Read more

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell-mediated in vivo control of HIV-1. Primary HIV-1-specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or "vertical" immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.

Rajapaksa US, Li D, Peng YC, McMichael AJ, Dong T, Xu XN. 2012. HLA-B may be more protective against HIV-1 than HLA-A because it resists negative regulatory factor (Nef) mediated down-regulation. Proc Natl Acad Sci U S A, 109 (33), pp. 13353-13358. | Show Abstract | Read more

Human leukocyte antigen HLA-B alleles have better protective activity against HIV-1 than HLA-A alleles, possibly due to differences in HLA-restricted HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) function, but the mechanism is unknown. HIV-1 negative regulatory factor (Nef) mediates down-regulation of surface expression of class I HLA (HLA-I) and may therefore impair immune recognition by CTL. Because of sequence differences in the cytoplasmic domains, HLA-A and -B are down-regulated by Nef but HLA-C and -E are not affected. However, the latter are expressed at low levels and are not of major importance in the CTL responses to HIV-1. Here, we compared the role of the cytoplasmic domains of HLA-A and -B in Nef-mediated escape from CTL. We found HLA-B cytoplasmic domains were more resistant to Nef-mediated down-regulation than HLA-A cytoplasmic domains and demonstrated that these differences affect CTL recognition of virus-infected cells in vitro. We propose that the relative resistance to Nef-mediated down-regulation by the cytoplasmic domains of HLA-B compared with HLA-A contributes to the better control of HIV-1 infection associated with HLA-B-restricted CTLs.

McMichael AJ, Borrow P, Tomaras GD, Goonetilleke N, Haynes BF. 2010. The immune response during acute HIV-1 infection: clues for vaccine development. Nat Rev Immunol, 10 (1), pp. 11-23. | Show Abstract | Read more

The early immune response to HIV-1 infection is likely to be an important factor in determining the clinical course of disease. Recent data indicate that the HIV-1 quasispecies that arise following a mucosal infection are usually derived from a single transmitted virus. Moreover, the finding that the first effective immune responses drive the selection of virus escape mutations provides insight into the earliest immune responses against the transmitted virus and their contributions to the control of acute viraemia. Strong innate and adaptive immune responses occur subsequently but they are too late to eliminate the infection. In this Review, we discuss recent studies on the kinetics and quality of early immune responses to HIV-1 and their implications for developing a successful preventive HIV-1 vaccine.

Goonetilleke N, Liu MK, Salazar-Gonzalez JF, Ferrari G, Giorgi E, Ganusov VV, Keele BF, Learn GH et al. 2009. The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection. J Exp Med, 206 (6), pp. 1253-1272. | Show Abstract | Read more

Identification of the transmitted/founder virus makes possible, for the first time, a genome-wide analysis of host immune responses against the infecting HIV-1 proteome. A complete dissection was made of the primary HIV-1-specific T cell response induced in three acutely infected patients. Cellular assays, together with new algorithms which identify sites of positive selection in the virus genome, showed that primary HIV-1-specific T cells rapidly select escape mutations concurrent with falling virus load in acute infection. Kinetic analysis and mathematical modeling of virus immune escape showed that the contribution of CD8 T cell-mediated killing of productively infected cells was earlier and much greater than previously recognized and that it contributed to the initial decline of plasma virus in acute infection. After virus escape, these first T cell responses often rapidly waned, leaving or being succeeded by T cell responses to epitopes which escaped more slowly or were invariant. These latter responses are likely to be important in maintaining the already established virus set point. In addition to mutations selected by T cells, there were other selected regions that accrued mutations more gradually but were not associated with a T cell response. These included clusters of mutations in envelope that were targeted by NAbs, a few isolated sites that reverted to the consensus sequence, and bystander mutations in linkage with T cell-driven escape.

Simmons A, Gangadharan B, Hodges A, Sharrocks K, Prabhakar S, García A, Dwek R, Zitzmann N, McMichael A. 2005. Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to positively regulate signaling. Immunity, 23 (6), pp. 621-634. | Show Abstract | Read more

Lentiviral Nef increases T cell signaling activity, but the molecular nature of the stimulus involved is incompletely described. We explored CD4 T cell lipid raft composition in the presence and absence of Nef. Here, the E2 ubiquitin-conjugating enzyme UbcH7, which acts in conjunction with c-Cbl, is absent from lipid rafts. This Nef-mediated exclusion is associated with failure of ubiquitination of activated Vav. In the presence of Nef, lipid raft Cdc42 is activated and forms a ternary complex between the c-Cbl-interacting protein p85Cool-1/betaPix and c-Cbl, displacing UbcH7 from rafts. Suppression of p85Cool-1/betaPix expression restores UbcH7 raft localization and Vav ubiquitination and diminishes Cdc42 activity. Moreover, p85Cool-1/betaPix knockdown attenuates HIV replication. Thresholds for activation of signaling involve the intricate balance of positive and negative regulators. Here we provide evidence for Nef disruption of a negative regulator of T cell signaling in promoting HIV replication.

Lee JK, Stewart-Jones G, Dong T, Harlos K, Di Gleria K, Dorrell L, Douek DC, van der Merwe PA, Jones EY, McMichael AJ. 2004. T cell cross-reactivity and conformational changes during TCR engagement. J Exp Med, 200 (11), pp. 1455-1466. | Show Abstract | Read more

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2-restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR-peptide-major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR-peptide-MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.

Ho LP, Urban BC, Jones L, Ogg GS, McMichael AJ. 2004. CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion. J Immunol, 172 (12), pp. 7350-7358. | Show Abstract

Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.

Stewart-Jones GB, McMichael AJ, Bell JI, Stuart DI, Jones EY. 2003. A structural basis for immunodominant human T cell receptor recognition. Nat Immunol, 4 (7), pp. 657-663. | Show Abstract | Read more

The anti-influenza CD8+ T cell response in HLA-A2-positive adults is almost exclusively directed at residues 58-66 of the virus matrix protein (MP(58-66)). V(beta)17V(alpha)10.2 T cell receptors (TCRs) containing a conserved arginine-serine-serine sequence in complementarity determining region 3 (CDR3) of the V(beta) segment dominate this response. To investigate the molecular basis of immunodominant selection in an outbred population, we have determined the crystal structure of V(beta)17V(alpha)10.2 in complex with MP(58-66)-HLA-A2 at a resolution of 1.4 A. We show that, whereas the TCR typically fits over an exposed side chain of the peptide, in this structure MP(58-66) exposes only main chain atoms. This distinctive orientation of V(beta)17V(alpha)10.2, which is almost orthogonal to the peptide-binding groove of HLA-A2, facilitates insertion of the conserved arginine in V(beta) CDR3 into a notch in the surface of MP(58-66)-HLA-A2. This previously unknown binding mode underlies the immunodominant T cell response.

Simmons A, Aluvihare V, McMichael A. 2001. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity, 14 (6), pp. 763-777. | Show Abstract | Read more

Gene expression profiling was used to explore the role of Nef in HIV. Nef induces a transcriptional program in T cells that is 97% identical to that of anti-CD3 T cell activation. This program is inhibited in the presence of cyclosporin. A requirement for TCR zeta and ZAP-70 is demonstrated for formation of the complete profile. Among eight factors particular to the anti-CD3 activation profile are IL16 and YY1, negative regulators of HIV transcription. In contrast, Nef exclusively upregulates factors positively regulating HIV, including Tat-SF1, U1 SNRNP, and IRF-2. New genes associated with Nef include CDK9, the induction of which enhances Tat function. Thus, Nef acts as a master switch early in the viral life cycle, forcing an environment conducive to dynamic viral production.

Kaul R, Dong T, Plummer FA, Kimani J, Rostron T, Kiama P, Njagi E, Irungu E et al. 2001. CD8(+) lymphocytes respond to different HIV epitopes in seronegative and infected subjects. J Clin Invest, 107 (10), pp. 1303-1310. | Show Abstract | Read more

HIV-1-specific cytotoxic T-lymphocyte (CTL) responses have been detected at a low frequency in many HIV-1-exposed, persistently seronegative (HEPS) subjects. However, it is unclear how CTLs could protect against HIV acquisition in HEPS subjects, when high levels of circulating CTL fail to prevent disease progression in most seropositive subjects. To address this issue we studied CD8(+) lymphocyte responses to a panel of HIV-1 CTL epitopes in 91 HEPS and 87 HIV-1-infected Nairobi sex workers. HIV-specific responses in seropositive women focused strongly on epitopes rarely or never recognized in HEPS subjects, who targeted epitopes that were subdominant or unrecognized in infected women. These differences in epitope specificity were restricted by only those HLA class I alleles that are associated with a reduced risk of HIV-1 infection in this cohort. Late seroconversion in HEPS donors was associated with a switch in epitope specificity and/or immunodominance to those epitopes preferentially recognized by HIV-1-infected women. The likelihood of detecting HIV-1-specific responses in HEPS women increased with the duration of viral exposure, suggesting that HIV-1-specific CD8(+) responses are acquired over time. The association between differential recognition of distinct CTL epitopes and protection from HIV-1 infection may have significant implications for vaccine design.

The dynamics of activating and inhibitory NK cell receptors at the cell-surface

Natural Killer (NK) cells are innate immune lymphocytes that rapidly destroy virally infected and malignant cells, and constitute major players in the early fight against disease.  Through ‘cross-talk’ with dendritic cells, monocytes and T lymphocytes, they also act as key immune modulators and influence long-term immunity in vivo.  NK cells primarily gauge abnormalities through the loss of cell-surface proteins known as MHC class I proteins on infected/tumour cells - this information is ...

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