Escape from HLA-B*08-restricted CD8 T cells by hepatitis C virus is associated with fitness costs

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

De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) evades CD8(+) T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8(+) T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8(+) T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8(+) T cells, four of which underwent mutation associated with dramatic loss of the original CD8(+) response. However, following the G(357)S escape in the HLA-A11-restricted Gag(349-359) epitope and the decline of wild-type-specific CD8(+) T-cell responses, a novel CD8(+) T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8(+) T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vbeta repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G(357)S escape variant of the Gag(349-359) epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8(+) T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8(+) T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.     

Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

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

Viral sequence evolution in acute hepatitis C virus infection

CD8(+)-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8+ -T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total non-envelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8+ T-cell responses. Interestingly, 19% of non-envelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8+ T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8+ T-cell responses and support the hypothesis that dysfunction of CD8+ T cells may be associated with failure to resolve HCV infections.     

The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr59-67) that is targeted by acute-phase HIV-1-specific CD8+ T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I60L variant all developed these responses. The I60L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.     

Immune responses induced in HHD mice by multiepitope HIV vaccine based on cryptic epitope modification

CD8+ T cells play an important role in early HIV infection. However, HIV has the capacity to avoid specific CTL responses due to a high rate of mutation under selection pressure. Although the HIV proteins, gag and pol, are relatively conserved, these sequences generate low-affinity MHC-associated epitopes that are poorly immunogenic. Here, we applied an approach that enhanced the immunogenicity of low-affinity HLA-A2.1-binding peptides. The first position with tyrosine (P1Y) substitution enhanced the affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. More importantly, P1Y variants efficiently stimulated in vivo native peptide-specific CTL that also recognized the corresponding naturally processed epitope. The potential to generate CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary technique for identification of virus cryptic epitopes for development of peptide-based immunotherapy. Therefore, identification and modification of the cryptic epitopes of gal and pol provides promising candidates for HIV immunotherapy dependent upon efficient presentation by virus cells. Furthermore, this may be a breakthrough that overcomes the obstacle of immune escape caused by high rates of mutation. In this study, bioinformatics analysis was used to predict six low-affinity cryptic HIV gag and pol epitopes presented by HLA-A*0201. A HIV compound multi-CTL epitope gene was constructed comprising the gene encoding the modified cryptic epitope and the HIV p24 antigen, which induced a strong CD8+ T cell immune response regardless of the mutation. This approach represents a novel strategy for the development of safe and effective HIV prophylactic and therapeutic vaccines.     

Escape from a dominant HLA-B*15-restricted CD8+ T cell response against hepatitis C virus requires compensatory mutations outside the epitope

Antiviral CD8(+) T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8(+) T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8(+) epitopes in NS5B (LLRHHNMVY(2450-2458) and SQRQKKVTF(2466-2474)), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.     

Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.     

In vivo hierarchy of immunodominant and subdominant HLA-A*0201-restricted T-cell epitopes of HBx antigen of hepatitis B virus

A polyepitopic CD8+ T-cell response is critical for the control of hepatitis B virus (HBV) infection. The HBV X protein (HBx) is a multifunctional protein that is important for the viral life cycle and for host-virus interactions. The aim of this study was to analyze the immunogenicity and dominance of various HLA-A*0201-restricted HBx-derived epitopes. For this purpose, we immunized HLA-A*0201-transgenic mice with HBx-derived peptides and DNA. This is a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, as these mice possess a cytotoxic T lymphocyte (CTL) repertoire representative of HLA-A2.1 individuals. We used cytotoxic tests and enzyme-linked immunosorbent spot (ELISPOT) assays to study the induction of specific cytotoxic and interferon (IFN)-gamma-secreting T cells. This allowed us to classify the HBx epitopes according to their T-cell activation capacity. After endogenous processing of the antigen synthesized in vivo after DNA-based immunization, we found that the HBx-specific T-cell response is targeted against one immunodominant epitope. Furthermore, following peptide immunization, we identified six additional novel subdominant T-cell epitopes. Inclusion of well-characterized epitopic sequences of HBx in a new vaccine for chronic HBV infections could help to broaden the T-cell response.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity

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

The antiviral efficacy of simian immunodeficiency virus-specific CD8+ T cells is unrelated to epitope specificity and is abrogated by viral escape

CD8(+) T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-gamma) enzyme-linked immunospot and IFN-gamma/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.     

Immunodomination in the evolution of dominant epitope-specific CD8+ T lymphocyte responses in simian immunodeficiency virus-infected rhesus monkeys

Because the control of HIV-1 replication is largely dependent on CD8+ T lymphocyte responses specific for immunodominant viral epitopes, vaccine strategies that increase the breadth of dominant epitope-specific responses should contribute to containing HIV-1 spread. Developing strategies to elicit such broad immune responses will require an understanding of the mechanisms responsible for focusing CD8+ T lymphocyte recognition on a limited number of epitopes. To explore this biology, we identified cohorts of rhesus monkeys that expressed the MHC class I molecules Mamu-A*01, Mamu-A*02, or both, and assessed the evolution of their dominant epitope-specific CD8+ T lymphocyte responses (Gag p11C- and Tat TL8-specific in the Mamu-A*01+ and Nef p199RY-specific in the Mamu-A*02+ monkeys) following acute SIV infection. The Mamu-A*02+ monkeys that also expressed Mamu-A*01 exhibited a significant delay in the evolution of the CD8+ T lymphocyte responses specific for the dominant Mamu-A*02-restricted SIV epitope, Nef p199RY. This delay in kinetics was not due to differences in viral load kinetics or magnitude or in viral escape mutations, but was associated with the evolution of the Mamu-A*01-restricted CD8+ T lymphocyte responses to the highly dominant SIV epitopes Gag p11C and Tat TL8. Thus, the evolution of dominant epitope-specific CD8+ T lymphocyte responses can be suppressed by other dominant epitope-specific responses, and this immunodomination is important in determining the kinetics of dominant epitope-specific responses.     

Dual pressure from antiretroviral therapy and cell-mediated immune response on the human immunodeficiency virus type 1 protease gene

Human immunodeficiency virus (HIV)-specific CD8(+) T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8(+) T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V(76-84) epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A(76-84) epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8(+) T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8(+) T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution.     

Immune Activation Promotes Evolutionary Conservation of T-Cell Epitopes in HIV-1

The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4⁺ helper T lymphocytes (T_H cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when T_H cell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved T_H epitopes may be counterproductive.     

Limited sequence evolution within persistently targeted CD8 epitopes in chronic human immunodeficiency virus type 1 infection

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-gamma) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-gamma responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.     

Identification of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine

Developing a peptide-based vaccine for the highly variable hepatitis C virus (HCV) remains a challenging task. Variant viruses not only escape antigen presentation but also persist in a patient as quasi-species. Such variants are often antagonistic to the responding T cell repertoire. To overcome these problems, we herein propose a cocktail vaccine consisting of a few epitope peptides, which make it possible to outpace the emergence of variant viruses. To design such a vaccine, we developed a way to identify HLA-A*2402-binding peptides efficiently by means of the computational scanning of the whole genome of the pathogen. Most of the predicted peptides exhibited strong binding to the HLA-A*2402 molecule, while also inducing CD8 T cell responses from the patients’ peripheral blood mononuclear cells (PBMCs). Peptide-induced T cells were capable of lysing HCV-expressing HepG2 cells which process antigens endogenously. The amount of HCV core antigen in the patients’ livers suggested that the lytic activity of the peptide-induced T cells was clearly in a range suitable for therapeutic use. If T cells were activated under optimal conditions by high density peptides, then they tended to be relatively tolerant of single amino acid variations for cytolysis. Finally, an analysis of the viral population isolated in Japan suggested no obvious changes due to immune evasion in the viral genome even in a host population highly biased toward HLA-A*2402. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

HLA-A*2402-restricted HIV-1-specific cytotoxic T lymphocytes and escape mutation after ART with structured treatment interruptions

Although a limited duration of immune activation of structured treatment interruptions (STIs) has been reported, the immune escape mechanism during STIs remains obscure. We therefore investigated the role of three immunodominant cytotoxic T lymphocyte (epitopes) in 12 HLA-A*2402-positive patients participating longitudinally during the clinical study of early antiretroviral treatment (ART) with five series of structured treatment interruptions (STIs). The frequency of HLA-A*2402-restricted CTLs varied widely and a sustained CTL response was rarely noted. However, a Y-to-F substitution at the second position in an immunodominant CTL epitope Nef138-10 (Nef138-2F), which was previously demonstrated as escape mutation, was frequently detected in seven patients primarily and emerged in the remaining five patients thereafter, and the existence of escape mutations was correlated with high pVL levels early in the clinical course. These findings suggest that escape mutation in the immunodominant CTL epitope may be one of the mechanisms to limit HIV-1-specific immune control in STIs.     

Anti-Hepatitis C Virus T-Cell Immunity in the Context of Multiple Exposures to the Virus

Characterisation of Hepatitis C virus (HCV)-specific CD8⁺ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8⁺ T-cell targets. Peptides representing these putative HCV CD8⁺ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design.     

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.