Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost

Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.     

Vaccination and timing influence SIV immune escape viral dynamics in vivo

CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help us understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods for following CTL escape and reversion are, however, insensitive to minor viral quasispecies. We developed sensitive quantitative real-time PCR assays to track the viral load of SIV Gag164-172 KP9 wild-type (WT) and escape mutant (EM) variants in pigtail macaques. Rapid outgrowth of EM virus occurs during the first few weeks of infection. However, the rate of escape plateaued soon after, revealing a prolonged persistence of WT viremia not detectable by standard cloning and sequencing methods. The rate of escape of KP9 correlated with levels of vaccine-primed KP9-specific CD8+ T cells present at that time. Similarly, when non-KP9 responder (lacking the restricting Mane-A*10 allele) macaques were infected with SHIVmn229 stock containing a mixture of EM and WT virus, rapid reversion to WT was observed over the first 2 weeks following infection. However, the rate of reversion to WT slowed dramatically over the first month of infection. The serial quantitation of escape mutant viruses evolving during SIV infection shows that rapid dynamics of immune escape and reversion can be observed in early infection, particularly when CD8 T cells are primed by vaccination. However, these early rapid rates of escape and reversion are transient and followed by a significant slowing in these rates later during infection, highlighting that the rate of escape is significantly influenced by the timing of its occurrence.     

Reversion of CTL escape-variant immunodeficiency viruses in vivo

Engendering cytotoxic T-lymphocyte (CTL) responses is likely to be an important goal of HIV vaccines. However, CTLs select for viral variants that escape immune detection. Maintenance of such escape variants in human populations could pose an obstacle to HIV vaccine development. We first observed that escape mutations in a heterogeneous simian immunodeficiency virus (SIV) isolate were lost upon passage to new animals. We therefore infected macaques with a cloned SIV bearing escape mutations in three immunodominant CTL epitopes, and followed viral evolution after infection. Here we show that each mutant epitope sequence continued to evolve in vivo, often re-establishing the original, CTL-susceptible sequence. We conclude that escape from CTL responses may exact a cost to viral fitness. In the absence of selective pressure upon transmission to new hosts, these original escape mutations can be lost. This suggests that some HIV CTL epitopes will be maintained in human populations.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

CD4+ target cell availability determines the dynamics of immune escape and reversion in vivo

Infections with human immunodeficiency virus (HIV) and the closely related monkey viruses simian-human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV) are characterized by progressive waves of immune responses, followed by viral mutation and "immune escape." However, escape mutation usually leads to lower replicative fitness, and in the absence of immune pressure, an escape mutant (EM) virus "reverts" to the wild-type phenotype. Analysis of the dynamics of immune escape and reversion has suggested it is a mechanism for identifying the immunogens best capable of controlling viremia. We have analyzed and modeled data of the dynamics of wild-type (WT) and EM viruses during SHIV infection of macaques. Modeling suggests that the dynamics of reversion and immune escape should be determined by the availability of target cells for infection. Consistent with this suggestion, we find that the rate of reversion of cytotoxic T-lymphocyte (CTL) EM virus strongly correlates with the number of CD4(+) T cells available for infection. This phenomenon also affects the rate of immune escape, since this rate is determined by the balance of CTL killing and the WT fitness advantage. This analysis predicts that the optimal timing for the selection of immune escape variants will be immediately after the peak of viremia and that the development of escape variants at later times will lead to slower selection. This has important implications for comparative studies of immune escape and reversion in different infections and for identifying epitopes with high fitness cost for use as vaccine targets.     

Reversion in vivo after inoculation of a molecular proviral DNA clone of simian immunodeficiency virus with a cytotoxic-T-lymphocyte escape mutation

Vaccine-based control of the replication of a simian immunodeficiency virus (SIV), SIVmac239, in macaques has recently been shown. In the process of the control, a mutant virus escaping from epitope-specific cytotoxic-T-lymphocyte (CTL) responses was rapidly selected and contained. In this study, we show that the wild-type virus appeared and became predominant in the absence of the epitope-specific CTL after inoculation of naive macaques with a molecular clone DNA of the CTL escape mutant SIV. This is the first report describing reversion in vivo from an inoculated, molecular proviral DNA clone of immunodeficiency virus with a CTL escape mutation.     

Tertiary Mutations Stabilize CD8+ T Lymphocyte Escape-Associated Compensatory Mutations following Transmission of Simian Immunodeficiency Virus

Compensatory mutations offset fitness defects resulting from CD8⁺ T lymphocyte (CD8_TL)-mediated escape, but their impact on viral evolution following transmission to naive hosts remains unclear. Here, we investigated the reversion kinetics of Gag_181–189CM9 CD8_TL escape-associated compensatory mutations in simian immunodeficiency virus (SIV)-infected macaques. Preexisting compensatory mutations did not result in acute-phase escape of the SIVmac239 CD8TL epitope Gag181-189CM9 and instead required a tertiary mutation for stabilization in the absence of Gag_181–189CM9 escape mutations. Therefore, transmitted compensatory mutations do not necessarily predict rapid CD8_TL escape.     

Evolution of human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitopes: fitness-balanced escape

CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.     

Fitness costs limit viral escape from cytotoxic T lymphocytes at a structurally constrained epitope

The intense selection pressure exerted by virus-specific cytotoxic T lymphocytes (CTL) on replicating human immunodeficiency virus and simian immunodeficiency virus results in the accumulation of CTL epitope mutations. It has been assumed that fitness costs can limit the evolution of CTL epitope mutations. However, only a limited number of studies have carefully examined this possibility. To explore the fitness costs associated with viral escape from p11C, C-M-specific CTL, we constructed a panel of viruses encoding point mutations at each position of the entire p11C, C-M epitope. Amino acid substitutions at positions 3, 4, 5, 6, 7, and 9 of the epitope significantly impaired virus replication by altering virus production and Gag protein expression as well as by destabilizing mature cores. Amino acid substitutions at position 2 of the epitope were tolerated but required reversion or additional compensatory mutations to generate replication-competent viruses. Finally, while amino acid substitutions at positions 1 and 8 of the p11C, C-M epitope were functionally tolerated, these substitutions were recognized by p11C, C-M-specific CTL and therefore provided no selection advantage for the virus. Together, these data suggest that limited sequence variation is tolerated by the region of the capsid encoding the p11C, C-M epitope and therefore that only a very limited number of mutations can allow successful viral escape from the p11C, C-M-specific CTL response.     

Rates of HIV immune escape and reversion: implications for vaccination

HIV-1 mutates extensively in vivo to escape immune control by CD8+ T cells (CTLs). The CTL escape mutant virus might also revert back to wild-type upon transmission to new hosts if significant fitness costs are incurred by the mutation. Immune escape and reversion can be extremely fast if they occur very early after infection, whereas they are much slower when they begin later during infection. Immune escape presents a significant barrier to vaccination, because escape of vaccine-mediated immune responses could neutralise any benefits of vaccination. Here, we consider the dynamics of immune escape and reversion in vivo in natural infection, and suggest how understanding of this can be used to predict optimal vaccine targets and design vaccination strategies that maximise immune control. We predict that inducing synchronous, broad CTL by vaccination should limit the likelihood of viral escape from immune control.     

The pigtail macaque MHC class I allele Mane-A*10 presents an immundominant SIV Gag epitope: identification, tetramer development and implications of immune escape and reversion

The pigtail macaque (Macaca nemestrina) is a common model for the study of AIDS. The pigtail major histocompatibility complex class I allele Mane-A*10 restricts an immunodominant simian immunodeficiency virus (SIV) Gag epitope (KP9) which rapidly mutates to escape T cell recognition following acute simian/human immunodeficiency virus infection. Two technologies for the detection of Mane-A*10 in outbred pigtail macaques were developed: reference strand-mediated conformational analysis and sequence-specific primer polymerase chain reaction. A Mane-A*10/KP9 tetramer was then developed to quantify CD8(+) T lymphocytes primed by multigenic DNA vaccination, which have previously been difficult to detect using standard interferon-gamma-based T cell assays. We also demonstrate mutational escape at KP9 following acute SIV infection. Mane-A*10(+) animals have lower set point SIV levels than Mane-A*10(-) animals, suggesting a significant fitness cost of escape. These studies pave the way for a more robust understanding of HIV vaccines in pigtail macaques.     

Involvement of multiple epitope-specific cytotoxic T-lymphocyte responses in vaccine-based control of simian immunodeficiency virus replication in rhesus macaques

Cytotoxic T-lymphocyte (CTL) responses are crucial for the control of immunodeficiency virus replication. Possible involvement of a dominant single epitope-specific CTL in control of viral replication has recently been indicated in preclinical AIDS vaccine trials, but it has remained unclear if multiple epitope-specific CTLs can be involved in the vaccine-based control. Here, by following up five rhesus macaques that showed vaccine-based control of primary replication of a simian immunodeficiency virus, SIVmac239, we present evidence indicating involvement of multiple epitope-specific CTL responses in this control. Three macaques maintained control for more than 2 years without additional mutations in the provirus. However, in the other two that shared a major histocompatibility complex haplotype, viral mutations were accumulated in a similar order, leading to viral evasion from three epitope-specific CTL responses with viral fitness costs. Accumulation of these multiple escape mutations resulted in the reappearance of plasma viremia around week 60 after challenge. Our results implicate multiple epitope-specific CTL responses in control of immunodeficiency virus replication and furthermore suggest that sequential accumulation of multiple CTL escape mutations, if allowed, can result in viral evasion from this control.     

Transmission of simian immunodeficiency virus carrying multiple cytotoxic T-lymphocyte escape mutations with diminished replicative ability can result in AIDS progression in rhesus macaques

Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations that result in escape from CTL recognition with viral fitness costs. The replication in vivo of such viruses carrying not single but multiple escape mutations in the absence of the CTL pressure has remained undetermined. Here, we have examined the replication of simian immunodeficiency virus (SIV) with five gag mutations selected in a macaque possessing the major histocompatibility complex haplotype 90-120-Ia after its transmission into 90-120-Ia-negative macaques. Our results showed that even such a "crippled" SIV infection can result in persistent viral replication, multiple reversions, and AIDS progression.     

Marked epitope- and allele-specific differences in rates of mutation in human immunodeficiency type 1 (HIV-1) Gag, Pol, and Nef cytotoxic T-lymphocyte epitopes in acute/early HIV-1 infection

During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in approximately 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.     

The distribution of CTL epitopes in HIV-1 appears to be random, and similar to that of other proteomes

Background  

HIV-1 viruses are highly capable of mutating their proteins to escape the presentation of CTL epitopes in their current host. Upon transmission to another host, some escape mutations revert, but other remain stable in the virus sequence for at least several years. Depending on the rate of accumulation and reversion of escape mutations, HIV-1 could reach a high level of adaptation to the human population. Yusim et. al. hypothesized that the apparent clustering of CTL epitopes in the conserved regions of HIV-1 proteins could be an evolutionary signature left by large-scale adaptation of HIV-1 to its human/simian host.     

Reversion and T Cell Escape Mutations Compensate the Fitness Loss of a CD8+ T Cell Escape Mutant in Their Cognate Transmitted/Founder Virus

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.     

Modelling the evolution and spread of HIV immune escape mutants

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

Vaccine-induced T cells control reversion of AIDS virus immune escape mutants

Many current-generation human immunodeficiency virus (HIV) vaccines induce specific T cells to control acute viremia, but their utility following infection with escape mutant virus is unclear. We studied reversion to wild type of an escape mutant simian-HIV in major histocompatibility complex-matched vaccinated pigtail macaques. High levels of vaccine-induced CD8+ T cells strongly correlated with maintenance of escape mutant virus during acute infection. Interestingly, in animals with lower CD8+ T-cell levels, transient reversion to wild-type virus resulted in better postacute control of viremia. Killing of wild-type virus facilitated by transient reversion outweighs the benefit of a larger CD8+ T-cell response that only maintains the less fit escape mutant virus. These findings have important implications for the further development of T-cell-based HIV vaccines where exposure to escape mutant viruses is common.     

Consequences of cytotoxic T-lymphocyte escape: common escape mutations in simian immunodeficiency virus are poorly recognized in naive hosts

Cytotoxic T lymphocytes (CTL) are associated with control of immunodeficiency virus infection but also select for variants that escape immune recognition. Declining frequencies of epitope-specific CTL frequencies have been correlated with viral escape in individual hosts. However, escape mutations may give rise to new epitopes that could be recognized by CTL expressing appropriate T-cell receptors and thus still be immunogenic when escape variants are passed to individuals expressing the appropriate major histocompatibility complex class I molecules. To determine whether peptide ligands that have been altered through escape can be immunogenic in new hosts, we challenged naïve, immunocompetent macaques with a molecularly cloned simian immunodeficiency virus (SIV) bearing common escape mutations in three immunodominant CTL epitopes. Responses to the altered peptides were barely detectable in fresh samples at any time after infection. Surprisingly, CTL specific for two of three escaped epitopes could be expanded by in vitro stimulation with synthetic peptides. Our results suggest that some escape variant epitopes evolving in infected individuals do not efficiently stimulate new populations of CTL, either in that individual or upon passage to new hosts. Nevertheless, escape variation may not completely abolish an epitope''s immunogenicity. Moreover, since the mutant epitope sequences did not revert to wild type during the study period, it is possible that low-frequency CTL exerted enough selective pressure to preserve epitope mutations in viruses replicating in vivo.In recent years, there has been increasing interest in AIDS vaccine approaches that elicit cytotoxic T lymphocytes (CTL), which recognize and eliminate cells infected with human immunodeficiency virus (HIV) (26). Unlike antibodies, effective CTL responses can be directed against epitopes derived from any viral protein, raising the possibility that CTL can be targeted to regions that are more conserved than the viral envelope. Current vaccine modalities can elicit potent CTL responses against multiple viral epitopes (25). Indeed, many lines of evidence indicate that cell-mediated immunity plays a major role in control of virus replication. Several studies have suggested an association between certain major histocompatibility complex (MHC) class I and class II alleles and control of viral replication or susceptibility to disease (6, 7, 11, 12, 15-17, 28, 36, 38, 39). CTL are also implicated in the initial control of immunodeficiency virus infection, since they appear in close temporal association with the reduction in peak viremia in both HIV-infected humans (5, 22) and simian immunodeficiency virus (SIV)-infected macaques (23). Antibody-mediated depletion of CD8⁺ cells in infected macaques resulted in dramatically increased virus loads in both acute and chronic infection (14, 27, 37).However, the plasticity of the viral genome also allows the generation of mutants that escape CTL recognition. Certain high-frequency CTL exert intense selective pressure on virus sequences, as revealed by the nearly total extinction of CTL-susceptible sequences from the actively replicating virus population within a few weeks of infection (2, 32). Escape from CTL has been observed in several studies of infected humans (12, 18, 21, 34, 35, 41) and macaques (2, 8, 30, 32, 40). Moreover, one report has shown that an HIV-1 escape mutant can be transmitted vertically (11), while other studies in vaccinated macaques have suggested that the evolution of escape mutants may be associated with a loss of containment of viral replication (4, 31). It therefore seems likely that escape from CTL responses occurs in most infected individuals (32).The apparent ubiquity of CTL escape may greatly complicate the design of CTL-based vaccines. The evolution of escape variants during infection of a single host may play a key part in viral persistence and therefore in the ultimate failure of immune containment and progression to AIDS. However, some investigators have suggested that T-cell receptor repertoires can recognize multiple epitope variants, so that CTL responses can coevolve along with viral escape variants in infected individuals (13). If T-cell receptor populations can recognize new variant epitopes arising within a single host, it seems plausible that variant epitope sequences could also be recognized efficiently in new hosts. Escape could also create “neo-epitopes,” novel sequences that are immunogenic to naïve T cells in individuals expressing the appropriate MHC class I molecules.The most rigorous test of the immunogenicity of epitopes altered through escape is to challenge a fully intact immune system with an escape mutant virus. Therefore, we identified common escape mutations that accumulated in immunodominant epitopes of SIVmac239 in infected macaques expressing the high-frequency MHC class I molecules Mamu-A*01 and Mamu-B*17. Together, these molecules bind three immunodominant CTL epitopes in SIVmac239: Gag_181-189CM9 (CTPYDINQM, Gag CM9) and Tat_28-35SL8 (STPESANL, Tat SL8) are bound by Mamu-A*01, and Nef_165-173IW9 (IRYPKTFGW, Nef IW9) is bound by Mamu-B*17. We have previously shown that the acute-phase response in Mamu-A*01 Mamu-B*17 double-positive macaques is dominated by CTL that recognize these three epitopes (33). We introduced common escape mutations into the SIVmac239 molecular clone and challenged macaques expressing both Mamu-A*01 and Mamu-B*17 with the mutant virus. CTL responses directed against the mutant epitopes were extremely low frequency or undetectable in fresh samples from each of the infected animals. In the absence of these responses, a completely new immunodominance hierarchy was established. Our results suggest it is unlikely that “escaped” epitopes will be recognized in newly infected individuals expressing appropriate MHC class I molecules.     

Functional constraints of influenza A virus epitopes limit escape from cytotoxic T lymphocytes

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.