Rational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A*02+ rhesus macaques with every SIV protein except Envelope (Env). Surprisingly, one of the strongest T cell responses engendered was against the Env protein, the Mamu-A*02–restricted epitope, Env788–795RY8. In this paper, we show that translation from an alternate reading frame of both the Rev-encoding DNA plasmid and the rAd5 vector engendered Env788–795RY8-specific CD8+ T cells of greater magnitude than “normal” SIV infection. Our data demonstrate both that the pathway from vaccination to immune response is not well understood and that products of alternate reading frames may be rich and untapped sources of T cell epitopes.

Vaccines designed to elicit AIDS virus-specific CD8؉ T cells should engender broad responses. Emerging data indicate that alternate reading frames (ARFs) of both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encode CD8 ؉ T cell epitopes, termed cryptic epitopes. Here, we show that SIV-specific CD8 ؉ T cells from SIV-infected rhesus macaques target 14 epitopes in eight ARFs during SIV infection. Animals recognized up to five epitopes, totaling nearly one-quarter of the anti-SIV responses. The epitopes were targeted by high-frequency responses as early as 2 weeks postinfection and in the chronic phase. Hence, previously overlooked ARF-encoded epitopes could be important components of AIDS vaccines.

Virus-specific CD8+ T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n=63 of 1240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIVmac239 for their capacity to bind Mamu-A1*007:01, 33 were found to bind with an affinity of 500nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T cell reactivity. In all, eleven of the peptides elicited IFN-γ+ T cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultra-deep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8+ T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8+ T cells and is part of the immune response to SIVmac239.

Understanding the correlates of immune protection against human immunodeficiency virus and simian immunodeficiency virus (SIV) will require defining the entire cellular immune response against the viruses. Here, we define two novel translation products from the SIV env mRNA that are targeted by the T-cell response in SIV-infected rhesus macaques. The shorter product is a subset of the larger product, which contains both the first exon of the Rev protein and a translated portion of the rev intron. Our data suggest that the translation of viral alternate reading frames may be an important source of T-cell epitopes, including epitopes normally derived from functional proteins.The pathway from viral infection to the cellular immune response is not well understood. Despite the importance of T-cell responses in control of AIDS virus replication (1,3,8,22), the sources of the peptides recognized by virus-specific T cells are still being discovered. AIDS virus-specific CD8 ϩ T lymphocytes (CD8-TL) recognize complexes of major histocompatibility complex (MHC) class I and virus-derived epitopes presented on the surface of infected cells. These epitopes can be derived from exogenous viral proteins in the infecting virion (19,20) or from de novo synthesis of viral proteins (9, 21). Additional sources of epitopes are also being explored (4, 6).CD8-TL can also recognize epitopes derived from translation of viral alternate reading frames (ARFs). Though CD8-TL specific for ARF-derived epitopes have been detected in human immunodeficiency virus (HIV) (2), they remain a largely unexplored source of epitopes that might elicit potent antiviral cellular immune responses. We recently showed that SIVmac239-infected rhesus macaques that spontaneously controlled viral replication, termed elite controllers, made immunodominant CD8-TL responses against an epitope (RHLAFK CLW, or cRW9) derived from an ARF of the env gene (15). This response selected for viral escape in vivo and suppressed viral replication in an in vitro assay. These findings imply that CD8-TL specific for ARF-derived epitopes might be an important component of the total AIDS virus-specific cellular immune response.Here, we show that the cRW9 epitope is translated as part of two distinct products that differ in size due to start codon usage. The larger and more frequent product contains both the first 23 amino acids of the Rev protein (exon 1) and 50 amino acids translated from the rev intron. The smaller is produced by translation initiation at a start codon within the rev intron and is a subset of the larger product. Finally, we show that these products are degraded after translation from the mature Envencoding mRNA.cRW9 is consistently recognized at 18 h postinfection. The extent of mRNA splicing is likely a critical determinant of the timing of both viral protein production and epitope recognition. The most rapidly recognized epitopes (excluding those derived from proteins present in the incoming virion) are those derived from proteins encoded by the fully spliced nef ...

Virus-specific CD8؉ T lymphocytes select for escape mutations in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). To assess the effects of these mutations on viral fitness, we introduced escape mutations into 30 epitopes (bound by five major histocompatibility complex class I [MHC-I] molecules) in three different viruses. Two of these MHC-I alleles are associated with elite control. Two of the three viruses demonstrated reduced fitness in vivo, and 27% of the introduced mutations reverted. These findings suggest that T cell epitope diversity may not be such a daunting problem for the development of an HIV vaccine.