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.