Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule,Mamu-A*02: a paradigm for virus evolution and persistence?

It is now accepted that an effective vaccine against AIDS must include effective cytotoxic-T-lymphocyte (CTL) responses. The simian immunodeficiency virus (SIV)-infected rhesus macaque is the best available animal model for AIDS, but analysis of macaque CTL responses has hitherto focused mainly on epitopes bound by a single major histocompatibility complex (MHC) class I molecule, Mamu-A*01. The availability of Mamu-A*01-positive macaques for vaccine studies is therefore severely limited. Furthermore, it is becoming clear that different CTL responses are able to control immunodeficiency virus replication with varying success, making it a priority to identify and analyze CTL responses restricted by common MHC class I molecules other than Mamu-A*01. Here we describe two novel epitopes derived from SIV, one from Gag (Gag(71-79) GY9), and one from the Nef protein (Nef(159-167) YY9). Both epitopes are bound by the common macaque MHC class I molecule, Mamu-A*02. The sequences of these two eptiopes are consistent with the molecule's peptide-binding motif, which we have defined by elution of natural ligands from Mamu-A*02. Strikingly, we found evidence for the selection of escape variant viruses by CTL specific for Nef(159-167) YY9 in 6 of 6 Mamu-A*02-positive animals. In contrast, viral sequences encoding the Gag(71-79) GY9 epitope remained intact in each animal. This situation is reminiscent of Mamu-A*01-restricted CTL that recognize Tat(28-35) SL8, which reproducibly selects for escape variants during acute infection, and Gag(181-189) CM9, which does not. Differential selection by CTL may therefore be a paradigm of immunodeficiency virus infection.     

The simian immunodeficiency virus envelope glycoprotein contains two epitopes presented by the Mamu-A*01 class I molecule.

Cytotoxic T lymphocyte (CTL) responses against the simian immunodeficiency virus (SIV) envelope and Gag proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC-->M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8(+) T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.     

Simian immunodeficiency virus evades a dominant epitope-specific cytotoxic T lymphocyte response through a mutation resulting in the accelerated dissociation of viral peptide and MHC class I

The ability of an AIDS virus to escape from immune containment by selective mutation away from recognition by CTL was explored in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. CTL recognition of a previously defined common viral mutation in an immunodominant SIVmac Gag epitope was evaluated. CTL were assessed for their ability to recognize a SIVmac Gag protein with a single residue 2 (T --> A) replacement in the minimal epitope peptide bound by the MHC class I molecule Mamu-A*01. SIVmac Gag-specific CTL lysed Mamu-A*01+ target cells infected with recombinant vaccinia virus expressing the wild-type but not the mutant Gag protein. In addition, CTL recognized the mutant epitope peptide less efficiently than the wild-type virus peptide. In studies to determine the mechanism by which the mutant virus evaded CTL recognition, this peptide was shown to bind Mamu-A*01 in a manner that was indistinguishable from the wild-type peptide. However, experiments in which an increasing duration of delay was introduced between peptide sensitization of target cells and the assessment of these cells as targets in killing assays suggest that the mutant peptide with a T --> A replacement had a higher off-rate from Mamu-A*01 than the wild-type peptide did. Therefore, these findings suggest that AIDS viruses can evade virus-specific CTL responses through the accelerated dissociation of mutant peptide from MHC class I.     

CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing

It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce a strong CD8(+) response. Additional desirable elements are multispecificity and a focus on conserved epitopes. The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means to achieve these goals. To test this concept in a relevant disease model we sought to identify multiple simian immunodeficiency virus (SIV)-derived CD8(+) epitopes bound by a single nonhuman primate major histocompatibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu-A*01(2), a common rhesus macaque MHC class I molecule that presents the immunodominant SIV gag-derived cytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for the presence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined using purified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent K(d) values of 500 nM or lower, with 21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infected Mamu-A*01(+) macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This study reveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents an important step toward the design of a multiepitope vaccine for SIV and HIV.     

Analysis of TCRalphabeta combinations used by simian immunodeficiency virus-specific CD8+ T cells in rhesus monkeys: implications for CTL immunodominance

Immunodominance is a common feature of Ag-specific CTL responses to infection or vaccines. Understanding the basis of immunodominance is crucial to understanding cellular immunity and viral evasion mechanisms and will provide a rational approach for improving HIV vaccine design. This study was performed comparing CTLs specific for the SIV Gag p11C (dominant) and SIV Pol p68A (subdominant) epitopes that are consistently generated in Mamu-A*01(+) rhesus monkeys exposed to SIV proteins. Additionally, vaccinated monkeys were used to prevent any issues of antigenic variation or dynamic changes in CTL responses by continuous Ag exposure. Analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-alpha- and -beta-chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of alpha- and beta-chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Our data suggest that Ag-specific naive T cell precursor frequency may be predetermined and that this process dictates immunodominance of SIV-specific CD8(+) T cell responses. These findings will aid in understanding immunodominance and designing new approaches to modulate CTL responses.     

Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.     

Immunodomination in the evolution of dominant epitope-specific CD8+ T lymphocyte responses in simian immunodeficiency virus-infected rhesus monkeys

Because the control of HIV-1 replication is largely dependent on CD8+ T lymphocyte responses specific for immunodominant viral epitopes, vaccine strategies that increase the breadth of dominant epitope-specific responses should contribute to containing HIV-1 spread. Developing strategies to elicit such broad immune responses will require an understanding of the mechanisms responsible for focusing CD8+ T lymphocyte recognition on a limited number of epitopes. To explore this biology, we identified cohorts of rhesus monkeys that expressed the MHC class I molecules Mamu-A*01, Mamu-A*02, or both, and assessed the evolution of their dominant epitope-specific CD8+ T lymphocyte responses (Gag p11C- and Tat TL8-specific in the Mamu-A*01+ and Nef p199RY-specific in the Mamu-A*02+ monkeys) following acute SIV infection. The Mamu-A*02+ monkeys that also expressed Mamu-A*01 exhibited a significant delay in the evolution of the CD8+ T lymphocyte responses specific for the dominant Mamu-A*02-restricted SIV epitope, Nef p199RY. This delay in kinetics was not due to differences in viral load kinetics or magnitude or in viral escape mutations, but was associated with the evolution of the Mamu-A*01-restricted CD8+ T lymphocyte responses to the highly dominant SIV epitopes Gag p11C and Tat TL8. Thus, the evolution of dominant epitope-specific CD8+ T lymphocyte responses can be suppressed by other dominant epitope-specific responses, and this immunodomination is important in determining the kinetics of dominant epitope-specific responses.     

Clonally diverse CTL response to a dominant viral epitope recognizes potential epitope variants.

RNA viruses undergo rapid sequence variation as the result of error-prone RNA replication mechanisms. When viable mutations arise in RNA regions encoding B or T cell epitopes, mutant viruses that can evade immune detection may be selected. In the carefully studied CTL response to the Gag p11C(C-M) epitope in SIVmac-infected Mamu-A*01(+) rhesus monkeys, it has been shown that CTL recognition of that epitope can occur even in the face of accruing mutations. To explore the underlying mechanism for this breadth of recognition, we have constructed Mamu-A*01 tetramers which discriminate T cells specific for epitope variants. Using these reagents we have defined discrete subsets of p11C(C-M)-specific T cells that cross-react with cells presenting variant peptides. We have found that individual Mamu-A*01(+) monkeys differ functionally in their ability to recognize epitope variants despite consistently strong recognition of the p11C(C-M) epitope. This functional difference is accounted for by the relative number of variant-specific T cells and by differences in the functionally relevant TCR repertoire of the infected monkeys. We have also found that monkeys immunized with DNA vaccine constructs encoding only the wild-type epitope sequence develop p11C(C-M)-specific CTL cross-reactive with variant peptides. Thus, cross-reactive CTL do not merely arise secondary to the emergence and immune presentation of viral CTL escape mutants but rather arise de novo following priming with a dominant epitope peptide sequence. Taken together, our results support the concept that the CTL response to a dominant viral epitope, although highly focused, can be clonally diverse and recognize potential epitope variants.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

Vaccine-elicited memory cytotoxic T lymphocytes contribute to Mamu-A*01-associated control of simian/human immunodeficiency virus 89.6P replication in rhesus monkeys

The expression of particular major histocompatibility complex (MHC) class I alleles can influence the rate of disease progression following lentiviral infections. This effect is a presumed consequence of potent cytotoxic T-lymphocyte (CTL) responses that are restricted by these MHC class I molecules. The present studies have examined the impact of the MHC class I allele Mamu-A*01 on simian/human immunodeficiency virus 89.6P (SHIV-89.6P) infection in unvaccinated and vaccinated rhesus monkeys by exploring the contribution of dominant-epitope specific CTL in this setting. Expression of Mamu-A*01 in immunologically naive monkeys was not associated with improved control of viral replication, CD4+ T-lymphocyte loss, or survival. In contrast, Mamu-A*01+ monkeys that had received heterologous prime/boost immunizations prior to challenge maintained higher CD4+ T-lymphocyte levels and better control of SHIV-89.6P replication than Mamu-A*01- monkeys. This protection was associated with the evolution of high-frequency anamnestic CTL responses specific for a dominant Mamu-A*01-restricted Gag epitope following infection. These data indicate that specific MHC class I alleles can confer protection in the setting of a pathogenic SHIV infection by their ability to elicit memory CTL following vaccination.     

Conserved MHC class I peptide binding motif between humans and rhesus macaques

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.     

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.     

Diverse peptide presentation of rhesus macaque major histocompatibility complex class I Mamu-A 02 revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus

Major histocompatibility complex class I (MHC I)-restricted CD8(+) T-cell responses play a pivotal role in anti-human immunodeficiency virus (HIV) immunity and the control of viremia. The rhesus macaque is an important animal model for HIV-related research. Among the MHC I alleles of the rhesus macaque, Mamu-A 02 is prevalent, presenting in ≥20% of macaques. In this study, we determined the crystal structure of Mamu-A 02, the second structure-determined MHC I from the rhesus macaque after Mamu-A 01. The peptide presentation characteristics of Mamu-A 02 are exhibited in complex structures with two typical Mamu-A 02-restricted CD8(+) T-cell epitopes, YY9 (Nef159 to -167; YTSGPGIRY) and GY9 (Gag71 to -79; GSENLKSLY), derived from simian immunodeficiency virus (SIV). These two peptides utilize similar primary anchor residues (Ser or Thr) at position 2 and Tyr at position 9. However, the central region of YY9 is different from that of GY9, a difference that may correlate with the immunogenic variance of these peptides. Further analysis indicated that the distinct conformations of these two peptides are modulated by four flexible residues in the Mamu-A 02 peptide-binding groove. The rare combination of these four residues in Mamu-A 02 leads to a variant presentation for peptides with different residues in their central regions. Additionally, in the two structures of the Mamu-A 02 complex, we compared the binding of rhesus and human β(2) microglobulin (β(2)m) to Mamu-A 02. We found that the peptide presentation of Mamu-A 02 is not affected by the interspecies interaction with human β(2)m. Our work broadens the understanding of CD8(+) T-cell-specific immunity against SIV in the rhesus macaque.     

The antiviral efficacy of simian immunodeficiency virus-specific CD8+ T cells is unrelated to epitope specificity and is abrogated by viral escape

CD8(+) T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-gamma) enzyme-linked immunospot and IFN-gamma/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.     

Contribution of CD8+ T cells to containment of viral replication and emergence of mutations in Mamu-A*01-restricted epitopes in Simian immunodeficiency virus-infected rhesus monkeys

Here, we investigated the containment of virus replication in simian immunodeficiency virus (SIV) infection by CD8(+) lymphocytes. Escape mutations in Mamu-A*01 epitopes appeared first in SIV Tat TL8 and then in SIV Gag p11C. The appearance of escape mutations in SIV Gag p11C was coincident with compensatory changes outside of the epitope. Eliminating CD8(+) lymphocytes from rhesus monkeys during primary infection resulted in more rapid disease progression that was associated with preservation of canonical epitopes. These results confirm the importance of cytotoxic T cells in controlling viremia and the constraint on epitope sequences that require compensatory changes to go to fixation.     

Major histocompatibility complex class I alleles associated with slow simian immunodeficiency virus disease progression bind epitopes recognized by dominant acute-phase cytotoxic-T-lymphocyte responses

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 SIV(mac)239 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 (Tat(28-35)SL8) and the Mamu-B*17-restricted response (Nef(165-173)IW9) typically select for viral escape variants in early SIV(mac)239 infection. Interestingly, animals expressing Mamu-A*1 and -B*17 have less variation in the Tat(28-35)SL8 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.     

Mucosal priming of simian immunodeficiency virus-specific cytotoxic T-lymphocyte responses in rhesus macaques by the Salmonella type III secretion antigen delivery system

Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested DeltaphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01(+) macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag(181-189) epitope were detected following each dose of SALMONELLA: After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag(181-189)-specific CD8(+) T-cell responses in peripheral blood. A significant percentage of the Gag(181-189)-specific T-cell population in each animal also expressed the intestinal homing receptor alpha4beta7. Additionally, Gag(181-189)-specific CD8(+) T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.     

Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge

The immunogenicity and protective efficacy of a modified vaccinia virus Ankara (MVA) recombinant expressing the simian immunodeficiency virus (SIV) Gag-Pol proteins (MVA-gag-pol) was explored in rhesus monkeys expressing the major histocompatibility complex (MHC) class I allele, MamuA*01. Macaques received four sequential intramuscular immunizations with the MVA-gag-pol recombinant virus or nonrecombinant MVA as a control. Gag-specific cytotoxic T-lymphocyte (CTL) responses were detected in all MVA-gag-pol-immunized macaques by both functional assays and flow cytometric analyses of CD8(+) T cells that bound a specific MHC complex class I-peptide tetramer, with levels peaking after the second immunization. Following challenge with uncloned SIVsmE660, all macaques became infected; however, viral load set points were lower in MVA-gag-pol-immunized macaques than in the MVA-immunized control macaques. MVA-gag-pol-immunized macaques exhibited a rapid and substantial anamnestic CTL response specific for the p11C, C-M Gag epitope. The level at which CTL stabilized after resolution of primary viremia correlated inversely with plasma viral load set point (P = 0.03). Most importantly, the magnitude of reduction in viremia in the vaccinees was predicted by the magnitude of the vaccine-elicited CTL response prior to SIV challenge.     

Identification of seventeen new simian immunodeficiency virus-derived CD8+ T cell epitopes restricted by the high frequency molecule, Mamu-A*02, and potential escape from CTL recognition

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 < or = 500 nM. We assessed the antigenicity of these 75 peptides using an IFN-gamma 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-gamma 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.