Persistent HIV-1-specific CTL clonal expansion despite high viral burden post in utero HIV-1 infection

To address the issue of clonal exhaustion in humans, we monitored HLA class I-restricted, epitope-specific CTL responses in an in utero HIV-1-infected infant from 3 mo through 5 years of age. Serial functional CTL precursor assays demonstrated persistent, vigorous, and broadly directed HIV-1 specific CTL activity with a dominant response against an epitope in HIV-1 Gag-p17 (SLYNTVATL, aa 77-85). A clonal CTL response directed against the immunodominant, HLA-A*0201-restricted epitope was found to persist over the entire observation period, as shown by TCR analysis of cDNA libraries generated from PBMC. The analysis of autologous viral sequences did not reveal any escape mutations within the targeted epitope, and viral load measurement indicated ongoing viral replication. Furthermore, inhibition of viral replication assays indicated that the epitope was properly processed from autologous viral protein. These data demonstrate that persistent exposure to high levels of viral Ag does not necessarily lead to clonal exhaustion and that epitope-specific clonal CTL responses induced within the first weeks of life can persist for years without inducing detectable viral escape variants.     

Cytotoxic T-cell antagonism in HIV-1

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

Impacts of avidity and specificity on the antiviral efficiency of HIV-1-specific CTL

Although CD8(+) CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.     

Dynamics of viral evolution and CTL responses in HIV-1 infection

Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection. We found that CTL responses developed sequentially and required constant antigenic stimulation for maintenance. CTL responses exerting strong selective pressure emerged early and led to rapid escape, proliferated rapidly and were predominant during acute/early infection. Although CTL responses to a few persistent epitopes developed over the first two months of infection, they proliferated slowly. As CTL epitopes were replaced by mutational variants, the corresponding responses immediately declined, most rapidly in the cases of strongly selected epitopes. CTL recognition of epitope variants, via cross-reactivity and de novo responses, was common throughout the period of study. Our data demonstrate that HIV-specific CTL responses, especially in the critical acute/early stage, were focused on regions that are prone to escape. Failure of CTL responses to strongly target functional or structurally critical regions of the virus, as well as the sequential cascade of CTL responses, followed closely by viral escape and decline of the corresponding responses, likely contribute to a lack of sustainable viral suppression. Focusing early and rapidly proliferating CTL on persistent epitopes may be essential for durable viral control in HIV-1 infection.     

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.     

CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.     

Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells.

A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.     

Candidate Vaccine Sequences to Represent Intra- and Inter-Clade HIV-1 Variation

A likely key factor in the failure of a HIV-1 vaccine based on cytotoxic T lymphocytes (CTL) is the natural immunodominance of epitopes that fall in variable regions of the proteome, which both increases the chance of epitope sequence mismatch with the incoming challenge strain and replicates the pathogenesis of early CTL failure due to epitope escape mutation during natural infection. To identify potential vaccine sequences to focus the CTL response on highly conserved epitopes, the whole proteomes of HIV-1 clades A1, B, C, and D were assessed for Shannon entropy at each amino acid position. Highly conserved regions in Gag (cGag-1, Gag 148–214, and cGag-2, Gag 253–331), Env (cEnv, Env 521–606), and Nef (cNef, Nef 106–148) were identified across clades. Inter- and intra-clade variability of amino acids within the regions tended to overlap, suggesting that polyvalent representation of consensus sequences for the four clades would allow broad HIV-1 strain representation. These four conserved regions were rich in both known and predicted CTL epitopes presented by a breadth of HLA types, and screening of 54 persons with chronic HIV-1 infection revealed that these regions are commonly immunogenic in the context of natural infection. These data suggest that vaccine delivery of a 16-valent mixture of these regions could focus the CTL response against conserved epitopes that are broadly representative of circulating HIV-1 strains.     

No evidence for competition between cytotoxic T-lymphocyte responses in HIV-1 infection

Strong competition between cytotoxic T-lymphocytes (CTLs) specific for different epitopes in human immunodeficiency virus (HIV) infection would have important implications for the design of an HIV vaccine. To investigate evidence for this type of competition, we analysed CTL response data from 97 patients with chronic HIV infection who were frequently sampled for up to 96 weeks. For each sample, CTL responses directed against a range of known epitopes in gag, pol and nef were measured using an enzyme-linked immunospot assay. The Lotka–Volterra model of competition was used to predict patterns that would be expected from these data if competitive interactions materially affect CTL numbers. In this application, the model predicts that when hosts make responses to a larger number of epitopes, they would have diminished responses to each epitope and that if one epitope-specific response becomes dramatically smaller, others would increase in size to compensate; conversely if one response grows, others would shrink. Analysis of the experimental data reveals results that are wholly inconsistent with these predictions. In hosts who respond to more epitopes, the average epitope-specific response tends to be larger, not smaller. Furthermore, responses to different epitopes almost always increase in unison or decrease in unison. Our findings are therefore inconsistent with the hypothesis that there is competition between CTL responses directed against different epitopes in HIV infection. This suggests that vaccines that elicit broad responses would be favourable because they would direct a larger total response against the virus, in addition to being more robust to the effects of CTL escape.     

Recognition of variant HIV-1 epitopes from diverse viral subtypes by vaccine-induced CTL

Recognition by CD8(+) T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.     

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.     

Sequential cleavage by metallopeptidases and proteasomes is involved in processing HIV-1 ENV epitope for endogenous MHC class I antigen presentation

Antigenic peptides derived from viral proteins by multiple proteolytic cleavages are bound by MHC class I molecules and recognized by CTL. Processing predominantly takes place in the cytosol of infected cells by the action of proteasomes. To identify other proteases involved in the endogenous generation of viral epitopes, specifically those derived from proteins routed to the secretory pathway, we investigated presentation of the HIV-1 ENV 10-mer epitope 318RGPGRAFVTI327 (p18) to specific CTL in the presence of diverse protease inhibitors. Both metalloproteinase and proteasome inhibitors decreased CTL recognition of the p18 epitope expressed from either native gp160 or from a chimera based on the hepatitis B virus secretory core protein as carrier protein. Processing of this epitope from both native ENV and the hepatitis B virus secretory core chimeric protein appeared to proceed by a TAP-dependent pathway that involved sequential cleavage by proteasomes and metallo-endopeptidases; however, other protease activities could replace the function of the lactacystin-sensitive proteasomes. By contrast, in a second TAP-independent pathway we detected no contribution of metallopeptidases for processing the ENV epitope from the chimeric protein. These results show that, in the classical TAP-dependent MHC class I pathway, endogenous Ag processing of viral proteins to yield the p18 10-mer epitope requires metallo-endopeptidases in addition to proteasomes.     

Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads

Background

HLA class-I alleles differ in their ability to control HIV replication through cell-mediated immune responses. No consistent associations have been found between the breadth of Cytotoxic T Lymphocytes (CTL) responses and the control of HIV-1, and it is unknown whether the size or distribution of the viral proteome-wide epitope repertoire, i.e., the intrinsic ability to present fewer, more or specific viral epitopes, could affect clinical markers of disease progression.

Methodology/Principal Findings

We used an epitope prediction model to identify all epitope motifs in a set of 302 HIV-1 full-length proteomes according to each individual''s HLA (Human Leukocyte Antigen) genotype. The epitope repertoire, i.e., the number of predicted epitopes per HIV-1 proteome, varied considerably between HLA alleles and thus among individual proteomes. In a subgroup of 270 chronically infected individuals, we found that lower viral loads and higher CD4 counts were associated with a larger predicted epitope repertoire. Additionally, in Gag and Rev only, more epitopes were restricted by alleles associated with low viral loads than by alleles associated with higher viral loads.

Conclusions/Significance

This comprehensive analysis puts forth the epitope repertoire as a mechanistic component of the multi-faceted HIV-specific CTL response. The favorable impact on markers of disease status of the propensity to present more HLA binding peptides and specific proteins gives impetus to vaccine design strategies that seek to elicit responses to a broad array of HIV-1 epitopes, and suggest a particular focus on Gag.     

The specificity and polymorphism of the MHC class I prevents the global adaptation of HIV-1 to the monomorphic proteasome and TAP

The large diversity in MHC class I molecules in a population lowers the chance that a virus infects a host to which it is pre-adapted to escape the MHC binding of CTL epitopes. However, viruses can also lose CTL epitopes by escaping the monomorphic antigen processing components of the pathway (proteasome and TAP) that create the epitope precursors. If viruses were to accumulate escape mutations affecting these monomorphic components, they would become pre-adapted to all hosts regardless of the MHC polymorphism. To assess whether viruses exploit this apparent vulnerability, we study the evolution of HIV-1 with bioinformatic tools that allow us to predict CTL epitopes, and quantify the frequency and accumulation of antigen processing escapes. We found that within hosts, proteasome and TAP escape mutations occur frequently. However, on the population level these escapes do not accumulate: the total number of predicted epitopes and epitope precursors in HIV-1 clade B has remained relatively constant over the last 30 years. We argue that this lack of adaptation can be explained by the combined effect of the MHC polymorphism and the high specificity of individual MHC molecules. Because of these two properties, only a subset of the epitope precursors in a host are potential epitopes, and that subset differs between hosts. We estimate that upon transmission of a virus to a new host 39%-66% of the mutations that caused epitope precursor escapes are released from immune selection pressure.