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.     

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.     

Estimating the effectiveness of simian immunodeficiency virus-specific CD8+ T cells from the dynamics of viral immune escape

Antiviral CD8(+) T cells are thought to play a significant role in limiting the viremia of human and simian immunodeficiency virus (HIV and SIV, respectively) infections. However, it has not been possible to measure the in vivo effectiveness of cytotoxic T cells (CTLs), and hence their contribution to the death rate of CD4(+) T cells is unknown. Here, we estimated the ability of a prototypic antigen-specific CTL response against a well-characterized epitope to recognize and kill infected target cells by monitoring the immunodominant Mamu-A*01-restricted Tat SL8 epitope for escape from Tat-specific CTLs in SIVmac239-infected macaques. Fitting a mathematical model that incorporates the temporal kinetics of specific CTLs to the frequency of Tat SL8 escape mutants during acute SIV infection allowed us to estimate the in vivo killing rate constant per Tat SL8-specific CTL. Using this unique data set, we show that at least during acute SIV infection, certain antiviral CD8(+) T cells can have a significant impact on shortening the longevity of infected CD4(+) T cells and hence on suppressing virus replication. Unfortunately, due to viral escape from immune pressure and a dependency of the effectiveness of antiviral CD8(+) T-cell responses on the availability of sufficient CD4(+) T cells, the impressive early potency of the CTL response may wane in the transition to the chronic stage of the 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.     

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.     

Comprehensive immunological evaluation reveals surprisingly few differences between elite controller and progressor Mamu-B*17-positive simian immunodeficiency virus-infected rhesus macaques

The association between particular major histocompatibility complex class I (MHC-I) alleles and control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication implies that certain CD8(+) T-lymphocyte (CD8-TL) responses are better able than others to control viral replication in vivo. However, possession of favorable alleles does not guarantee improved prognosis or viral control. In rhesus macaques, the MHC-I allele Mamu-B*17 is correlated with reduced viremia and is overrepresented in macaques that control SIVmac239, termed elite controllers (ECs). However, there is so far no mechanistic explanation for this phenomenon. Here we show that the chronic-phase Mamu-B*17-restricted repertoire is focused primarily against just five epitopes-VifHW8, EnvFW9, NefIW9, NefMW9, and env(ARF)cRW9-in both ECs and progressors. Interestingly, Mamu-B*17-restricted CD8-TL do not target epitopes in Gag. CD8-TL escape variation occurred in all targeted Mamu-B*17-restricted epitopes. However, recognition of escape variant peptides was commonly observed in both ECs and progressors. Wild-type sequences in the VifHW8 epitope tended to be conserved in ECs, but there was no evidence that this enhances viral control. In fact, no consistent differences were detected between ECs and progressors in any measured parameter. Our data suggest that the narrowly focused Mamu-B*17-restricted repertoire suppresses virus replication and drives viral evolution. It is, however, insufficient in the majority of individuals that express the "protective" Mamu-B*17 molecule. Most importantly, our data indicate that the important differences between Mamu-B*17-positive ECs and progressors are not readily discernible using standard assays to measure immune responses.     

Chinese origin rhesus macaque major histocompatibility complex class I molecules promiscuously present epitopes from SIV associated with molecules of Indian origin; implications for immunodominance and viral escape

The presentation of identical peptides by different major histocompatibility complex class I (MHC-I) molecules, termed promiscuity, is a controversial feature of T cell-mediated immunity to pathogens. The astounding diversity of MHC-I molecules in human populations, presumably to enable binding of equally diverse peptides, implies promiscuity would be a rare phenomenon. However, if it occurs, it would have important implications for immunity. We screened 77 animals for responses to peptides known to bind MHC-I molecules that were not expressed by these animals. Some cases of supposed promiscuity were determined to be the result of either non-identical optimal peptides or were simply not mapped to the correct MHC-I molecule in previous studies. Cases of promiscuity, however, were associated with alterations of immunodominance hierarchies, either in terms of the repertoire of peptides presented by the different MHC-I molecules or in the magnitude of the responses directed against the epitopes themselves. Specifically, we found that the Mamu-B*017:01-restricted peptides Vif HW8 and cRW9 were also presented by Mamu-A2*05:26 and targeted by an animal expressing that allele. We also found that the normally subdominant Mamu-A1*001:01 presented peptide Gag QI9 was also presented by Mamu-B*056:01. Both A2*05:26 and B*056:01 are molecules typically or exclusively expressed by animals of Chinese origin. These data clearly demonstrate that MHC-I epitope promiscuity, though rare, might have important implications for immunodominance and for the transmission of escape mutations, depending on the relative frequencies of the given alleles in a population.     

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.     

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.     

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency.

T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIV(mac251 (561)). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIV(mac251 (561)) or SIV(SME660). However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV(89.6P) or SHIV(KU2), respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.     

Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression

Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.     

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.     

Characterization of the peptide-binding specificity of Mamu-B*17 and identification of Mamu-B*17-restricted epitopes derived from simian immunodeficiency virus proteins

The SIV-infected rhesus macaque is an excellent model to examine candidate AIDS virus vaccines. These vaccines should elicit strong CD8(+) responses. Previous definition of the peptide-binding motif and optimal peptides for Mamu-A*01 has created a demand for Mamu-A*01-positive animals. We have now studied a second MHC class I molecule, Mamu-B*17, that is present in 12% of captive-bred Indian rhesus macaques. The peptide-binding specificity of the Mamu-B*17 molecule was characterized using single substitution analogs of two Mamu-B*17-binding peptides and libraries of naturally occurring sequences of viral or bacterial origin. Mamu-B*17 uses position 2 and the C terminus of its peptide ligands as dominant anchor residues. The C terminus was found to have a very narrow specificity for the bulky aromatic residue W, with other aromatic residues (F and Y) being only occasionally tolerated. Position 2 is associated with a broad chemical specificity, readily accommodating basic (H and R), bulky hydrophobic (F and M), and small aliphatic (A) residues. Using this motif, we identified 50 peptides derived from SIV(mac)239 that bound Mamu-B*17 with an affinity of 500 nM or better. ELISPOT and intracellular cytokine-staining assays showed that 16 of these peptides were antigenic. We have, therefore, doubled the number of MHC class I molecules for which SIV-derived binding peptides have been characterized. This allows for the quantitation of immune responses through tetramers and analysis of CD8(+) function by intracellular cytokine-staining assays and ELISPOT. Furthermore, it is an important step toward the design of a multiepitope vaccine for SIV and HIV.     

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.     

Macaque long-term nonprogressors resist superinfection with multiple CD8+ T cell escape variants of simian immunodeficiency virus

Human immunodeficiency virus (HIV)-positive individuals can be superinfected with different virus strains. Individuals who control an initial HIV infection are therefore still at risk for subsequent infection with divergent viruses, but the barriers to such superinfection remain unclear. Here we tested long-term nonprogressors' (LTNPs') susceptibility to superinfection using Indian rhesus macaques that express the major histocompatibility complex class I (MHC-I) allele Mamu-B 17, which is associated with control of the pathogenic AIDS virus SIVmac239. The Mamu-B 17-restricted CD8(+) T cell repertoire is focused almost entirely on 5 epitopes. We engineered a series of SIVmac239 variants bearing mutations in 3, 4, or all 5 of these epitopes and used them to serially challenge 2 Mamu-B 17-positive LTNPs. None of the escape variants caused breakthrough replication in LTNPs, although they readily infected Mamu-B 17-negative naive macaques. In vitro competing coculture assays and examination of viral evolution in hosts lacking Mamu-B 17 suggested that the mutant viruses had negligible defects in replicative fitness. Both LTNPs maintained robust immune responses, including simian immunodeficiency virus (SIV)-specific CD8(+) and CD4(+) T cells and neutralizing antibodies. Our results suggest that escape mutations in epitopes bound by "protective" MHC-I molecules may not be sufficient to establish superinfection in LTNPs.     

Expression of the major histocompatibility complex class I molecule Mamu-A*01 is associated with control of simian immunodeficiency virus SIVmac239 replication

Several HLA alleles are associated with attenuated human immunodeficiency virus disease progression. We explored the relationship between the expression of particular major histocompatibility complex (MHC) class I alleles and viremia in simian immunodeficiency virus SIV(mac)239-infected macaques. Of the common MHC class I alleles, animals that expressed Mamu-A*01 exhibited the best control of viral replication.     

Immunization of macaques with single-cycle simian immunodeficiency virus (SIV) stimulates diverse virus-specific immune responses and reduces viral loads after challenge with SIVmac239

Genetically engineered simian immunodeficiency viruses (SIV) that is limited to a single cycle of infection was evaluated as a nonreplicating AIDS vaccine approach for rhesus macaques. Four Mamu-A*01(+) macaques were inoculated intravenously with three concentrated doses of single-cycle SIV (scSIV). Each dose consisted of a mixture of approximately equivalent amounts of scSIV strains expressing the SIV(mac)239 and SIV(mac)316 envelope glycoproteins with mutations in nef that prevent major histocompatibility complex (MHC) class I downregulation. Viral loads in plasma peaked between 10(4) and 10(5) RNA copies/ml on day 4 after the first inoculation and then steadily declined to undetectable levels over the next 4 weeks. SIV Gag-specific T-cell responses were detected in peripheral blood by MHC class I tetramer staining (peak, 0.07 to 0.2% CD8(+) T cells at week 2) and gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assays (peak, 50 to 250 spot forming cells/10(6) peripheral blood mononuclear cell at week 3). Following the second and third inoculations at weeks 8 and 33, respectively, viral loads in plasma peaked between 10(2) and 10(4) RNA copies/ml on day 2 and were cleared over a 1-week period. T-cell-proliferative responses and antibodies to SIV were also observed after the second inoculation. Six weeks after the third dose, each animal was challenged intravenously with SIV(mac)239. All four animals became infected. However, three of the four scSIV-immunized animals exhibited 1 to 3 log reductions in acute-phase plasma viral loads relative to two Mamu-A*01(+) control animals. Additionally, two of these animals were able to contain their viral loads below 2,000 RNA copies/ml as late as 35 weeks into the chronic phase of infection. Given the extraordinary difficulty in protecting against SIV(mac)239, these results are encouraging and support further evaluation of lentiviruses that are limited to a single cycle of infection as a preclinical AIDS vaccine approach.     

The role of MHC class I allele Mamu-A*07 during SIVmac239 infection

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 1,240). 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 SIV(mac)239 for their capacity to bind Mamu-A1*007:01; 33 were found to bind with an affinity of 500?nM 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, 11 of the peptides elicited IFN-γ(+) T-cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultradeep 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 SIV(mac)239.     

Mamu-A*01 allele-mediated attenuation of disease progression in simian-human immunodeficiency virus infection

Expression of several major histocompatibility complex (MHC) class I alleles is associated with a protective effect against disease progression in both human immunodeficiency virus type 1 and simian immunodeficiency virus infection. To understand the mechanism underlying this effect, we investigated the expression of the MHC class I allele Mamu-A*01 in simian-human immunodeficiency virus (SHIV) infection, one of the major models for evaluation of AIDS vaccine candidates. We found that disease progression was significantly delayed in Mamu-A*01-positive rhesus monkeys infected with the highly pathogenic SHIV 89.6P. The delay corresponded not only to a noted Mamu-A*01-restricted dominant cytotoxic T-lymphocyte (CTL) response but also to a lower viral load in lymph nodes (LN) and, importantly, to minimal destruction of LN structure during early infection. In contrast, Mamu-A*01-negative monkeys exhibited massive destruction of LN structure with accompanying rapid disease progression. These data indicate that MHC class I allele-restricted CTL responses may play an important role in preservation of lymphoid tissue structure, thereby resulting in attenuation of disease progression in immunodeficiency virus infection.