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.

Cytotoxic T-lymphocyte (CTL) responses peak coincident with the decline in acute HIV viremia. Despite two reports of CTL-resistant HIV variants emerging during acute infection, the contribution of acute CTL escape to HIV pathogenesis remains unclear. Difficulties inherent in studying acute HIV infection can be overcome by modeling virus-host interactions in SIV-infected rhesus macaques. We sequenced 21 complete simian immunodeficiency virus (SIV)mac239 genomes at four weeks post-infection to determine the extent of acute CTL escape. Here we show that viruses from 19 of 21 macaques escaped from CTLs during acute infection and that these escape-selecting CTLs were responsive to lower concentrations of peptide than other SIV-specific CTLs. Interestingly, CTLs that require low peptide concentrations for stimulation (high 'functional avidity') are particularly effective at controlling other viral infections. Our results suggest that acute viral escape from CTLs is a hallmark of SIV infection and that CTLs with high functional avidity can rapidly select for escape variants.

Certain major histocompatibility complex class I (MHC-I) alleles are associated with delayed disease progression in individuals infected with human immunodeficiency virus (HIV) and in macaques infected with simian immunodeficiency virus (SIV). However, little is known about the influence of these MHC alleles on acute-phase cellular immune responses. Here we follow 51 animals infected with SIVmac239 and demonstrate a dramatic association between Mamu-A*01 and -B*17 expression and slowed disease progression. We show that the dominant acute-phase cytotoxic T lymphocyte (CTL) responses in animals expressing these alleles are largely directed against two epitopes restricted by Mamu-A*01 and one epitope restricted by Mamu-B*17. One Mamu-A*01-restricted response (Tat28-35SL8) and the Mamu-B*17-restricted response (Nef165-173IW9) typically select for viral escape variants in early SIVmac239 infection. Interestingly, animals expressing Mamu-A*1 and -B*17 have less variation in the Tat28-35SL8 epitope during chronic infection than animals that express only Mamu-A*01. Our results show that MHC-I alleles that are associated with slow progression to AIDS bind epitopes recognized by dominant CTL responses during acute infection and underscore the importance of understanding CTL responses during primary HIV infection

It is now accepted that an effective vaccine against AIDS must include effective cytotoxic-T-lymphocyte (CTL) responses. The simian immunodeficiency virus (SIV)-infected rhesusAs the search for a safe and effective vaccine against AIDS enters its third decade, the pandemic continues, with over 40 million human immunodeficiency virus (HIV)-infected individuals worldwide as of the end of 2001 (36). The need for a prophylactic vaccine, and thus for a clear understanding of effective antiviral immune responses, is increasing in urgency.

MHC class I-restricted CD8+ T cells play an important role in controlling HIV and SIV replication. In SIV-infected Indian rhesus macaques (Macaca mulatta), comprehensive CD8+ T cell epitope identification has only been undertaken for two alleles, Mamu-A*01 and Mamu-B*17. As a result, these two molecules account for virtually all known MHC class I-restricted SIV-derived CD8+ T cell epitopes. SIV pathogenesis research and vaccine testing have intensified the demand for epitopes restricted by additional MHC class I alleles due to the shortage of Mamu-A*01+ animals. Mamu-A*02 is a high frequency allele present in over 20% of macaques. In this study, we characterized the peptide binding of Mamu-A*02 using a panel of single amino acid substitution analogues and a library of 497 unrelated peptides. Of 230 SIVmac239 peptides that fit the Mamu-A*02 peptide-binding motif, 75 peptides bound Mamu-A*02 with IC50 values of ≤500 nM. We assessed the antigenicity of these 75 peptides using an IFN-γ ELISPOT assay with freshly isolated PBMC from eight Mamu-A*02+ SIV-infected macaques and identified 17 new epitopes for Mamu-A*02. The synthesis of five Mamu-A*02 tetramers demonstrated the discrepancy between tetramer binding and IFN-γ secretion by SIV-specific CD8+ T cells during chronic SIV infection. Bulk sequencing determined that 2 of the 17 epitopes accumulated amino acid replacements in SIV-infected macaques by the chronic phase of infection, suggestive of CD8+ T cell escape in vivo. This work enhances the use of the SIV-infected macaque model for HIV and increases our understanding of the breadth of CD8+ T cell responses in SIV infection.

Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in gag in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.

CD8؉ T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope. CD8ϩ -T-lymphocyte (CD8-TL) responses against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) select viral escape variants that are poorly recognized by the immune system. The number of CD8-TL responses that select escape variants and the contribution of CD8-TL selection to viral diversification are unknown. CD8-TL responses against HIV and SIV are broadly directed, as they target epitopes in all viral proteins (11,26). If many of these responses select for viral variants, CD8-TL escape may be principally responsible for sequence changes that accumulate within infected individuals, an outcome recently hypothesized by Overbaugh and Bangham (40).Until recently, the molecular and immunologic tools that are necessary to analyze CD8-TL selection at the genome level have been unavailable. Most studies of CD8-TL escape have focused on single epitopes or small groups of epitopes clustered in single SIV and HIV genes (4,7,16,20,22,43,44). While they were instrumental in demonstrating that CD8-TL selection is a cardinal feature of SIV and HIV infections, these studies could not comprehensively evaluate the frequency of CD8-TL escape throughout the viral genome.High-throughput DNA sequencing technologies now enable the sequencing of entire SIV genomes from infected animals (38). Additionally, 46 CD8-TL epitopes bound by the highfrequency Indian rhesus macaque major histocompatibility complex class I (MHC class I) alleles Mamu-A‫10ء‬ (3), -A‫20ء‬ (28a), and -B‫71ء‬ (34) are now known. By sequencing complete viral genomes spanning these 46 CD8-TL epitopes, we can now directly measure the frequency of CD8-TL selection.With this study, we found that CD8-TL escape is common, with sequences encoding 23 of 46 epitopes accumulating variations that are consistent with escape. Within a single animal, Ͼ64% of the nonsynonymous viral variants outside of the envelope were located within recognized CD8-TL epitopes, suggesting that CD8-TL escape is a primary selector of withinhost diversity. MATERIALS AND METHODSViruses and animals. The 35 animals used for the bulk of the analyses were infected with SIVmac239 between 1998 and 2001. These animals were challenged with SIV in trials of vaccine efficacy and pathogenesis...

No abstract