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.     

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.     

Efficient class I major histocompatibility complex down-regulation by simian immunodeficiency virus Nef is associated with a strong selective advantage in infected rhesus macaques

Substitution of Y223F disrupts the ability of simian immunodeficiency virus (SIV) Nef to down-modulate major histocompatibility complex (MHC) class I from the cell surface but has no effect on other Nef functions, such as down-regulation of CD4, CD28, and CD3 cell surface expression or stimulation of viral replication and enhancement of virion infectivity. Inoculation of three rhesus macaques with the SIVmac239 Y223F-Nef variant revealed that this point mutation consistently reverts and that Nef activity in MHC class I down-modulation is fully restored within 4 weeks after infection. Our results demonstrate a strong selective pressure for a tyrosine at amino acid position 223 in SIV Nef, and they constitute evidence that Nef-mediated MHC class I down-regulation provides a selective advantage for viral replication in vivo.     

Selective downregulation of rhesus macaque and sooty mangabey major histocompatibility complex class I molecules by Nef alleles of simian immunodeficiency virus and human immunodeficiency virus type 2

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIV(smm/mac) and HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys.     

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.     

Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys.

Infection of macaque monkeys with the simian immunodeficiency virus of macaques (SIVmac) results in disease similar to human AIDS. Therefore, the macaque monkey is proving to be an important model for testing the effectiveness of various AIDS vaccine approaches. A detailed analysis of the cellular immune responses is necessary for the evaluation of candidate vaccines. However, this has not been possible in macaques, due, in part, to the unknown nature of the MHC molecules that restrict their T lymphocytes. In our report we demonstrate that a particular MHC class I molecule involved in the rhesus monkey's effector T lymphocyte response to SIVmac is expressed at a high frequency in a colony of rhesus monkeys. SIVmac-infected monkeys that express this MHC class I molecule all develop CTL that are restricted by that molecule and recognize an identical nine amino acid epitope of the SIVmac gag protein. This MHC class I molecule has been defined as an HLA-A homolog by cDNA cloning and sequencing. It has also been expressed in an MHC class I-deficient cell line to demonstrate directly the cloned molecule's capacity to bind and present peptide Ag to CTL. These studies illustrate that AIDS virus-specific CTL can be characterized in detail in the rhesus monkey and lay the foundation for exploring novel approaches to AIDS virus vaccination in this species.     

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.     

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.     

恒河猴MHC-I类分子Mamu-A*02与猴免疫缺陷病毒抗原表位复合物的纯化和晶体学分析

恒河猴是研究人免疫缺陷型病毒(HIV)感染与获得性免疫缺失综合征(AIDS)疫苗研制的最为重要的动物模型,其组织相容性复合体(MHC)结构的解析将有助于了解HIV免疫逃逸机制。本研究克隆了1个恒河猴MHC-I类分子Mamu-A*02轻链(β2m)基因并插入到pET21a(+)原核表达载体中,成功构建了重组表达质粒pET21a(+)-Mamu-β2m。pET21a(+)-Mamu-β2m和之前构建好的恒河猴Mamu-A*02重链(α)重组质粒pET21a(+)-α分别转入BL21(DE3)大肠杆菌表达体系中,IPTG诱导表达。Mamu-A*02重链和猴β2m目的蛋白在BL21(DE3)中以包涵体形式表达。重链、轻链的包涵体蛋白和Mamu-A*02限制的猴免疫缺陷病毒(SIV)Nef表位多肽(YY9)通过稀释法共复性。高纯度的复合物蛋白经分子筛层析和阴离子交换层析纯化后获得。复合物晶体利用悬滴法筛选并优化,并在0.1mol/LBIS-TRIS(pH5.5)、2.0mol/L(NH4)2SO4条件下收集一套2.8?分辨率的X-射线衍射数据。晶体属于正交空间群P212121,其晶胞参数为a=128.99?,b=129...     

The high-frequency major histocompatibility complex class I allele Mamu-B*17 is associated with control of simian immunodeficiency virus SIVmac239 replication

Particular HLA alleles are associated with reduced human immunodeficiency virus replication. It has been difficult, however, to characterize the immune correlates of viral control. An analysis of the influence of major histocompatibility complex class I alleles on viral control in 181 simian immunodeficiency virus SIVmac239-infected rhesus macaques revealed that Mamu-B(*)17 was associated with a 26-fold reduction in plasma virus concentrations (P<0.001). Mamu-B(*)17 was also enriched in a group of animals that controlled viral replication by 1,000-fold [corrected] Even after accounting for this group, Mamu-B(*)17 was associated with an eightfold reduction in plasma virus concentrations (P<0.001). Mamu-B(*)17-positive macaques could, therefore, facilitate our understanding of the correlates of viral control.     

Detection of simian immunodeficiency virus Gag-specific CD8(+) T lymphocytes in semen of chronically infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex

Evaluation of human immunodeficiency virus type 1-specific mucosal cytotoxic T lymphocytes can be hampered by limited cell yields from mucosal sites. We sought to characterize virus-specific CD8(+) T lymphocytes with cytotoxic activity in the male genital tracts of SIVmac-infected rhesus monkeys by using a peptide epitope-specific functional T-cell assay and a tetrameric major histocompatibility complex class I-peptide complex. This tetrameric complex was constructed with the rhesus monkey HLA-A homolog molecule Mamu-A*01 and a dominant-epitope 9-amino-acid fragment of SIVmac Gag (p11C, C-M). The proportion of tetramer-positive CD8(+) T cells in semen of SIVmac-infected monkeys ranged from 5.9 to 22.0%. By the use of a standard 51Cr release assay, these cells were found to have peptide epitope-specific cytolytic activity after in vitro expansion. Four-color flow-cytometric analysis of these seminal tetramer-positive CD8(+) T cells demonstrated that they express memory-associated (CD62L- CD45RA-) and activation-associated (CD11a+ Fas+ HLA-DR+) molecules. The present experiments illustrate the power of tetramer technology for evaluating antigen-specific CD8(+) T lymphocytes in a mucosal tissue compartment.     

Major histocompatibility complex class I-restricted cytotoxic T lymphocyte responses during primary simian immunodeficiency virus infection in Burmese rhesus macaques

Major histocompatibility complex class I (MHC-I)-restricted CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. In particular, Gag-specific CTL responses have been shown to exert strong suppressive pressure on HIV/SIV replication. Additionally, association of Vif-specific CTL frequencies with in vitro anti-SIV efficacy has been suggested recently. Host MHC-I genotypes could affect the immunodominance patterns of these potent CTL responses. Here, Gag- and Vif-specific CTL responses during primary SIVmac239 infection were examined in three groups of Burmese rhesus macaques, each group having a different MHC-I haplotype. The first group of four macaques, which possessed the MHC-I haplotype 90-010-Ie, did not show Gag- or Vif-specific CTL responses. However, Nef-specific CTL responses were elicited, suggesting that primary SIV infection does not induce predominant CTL responses specific for Gag/Vif epitopes restricted by 90-010-Ie-derived MHC-I molecules. In contrast, Gag- and Vif-specific CTL responses were induced in the second group of two 89-075-Iw-positive animals and the third group of two 91-010-Is-positive animals. Considering the potential of prophylactic vaccination to affect CTL immunodominance post-viral exposure, these groups of macaques would be useful for evaluation of vaccine antigen-specific CTL efficacy against SIV infection.     

The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques

The simian immunodeficiency virus of macaques (SIVmac) is a lentivirus which induces an AIDS-like disease in rhesus monkeys. We have explored the virus-specific cellular immune response in SIVmac-infected rhesus monkeys. Con A-activated, IL-2 expanded PBL of some SIVmac-infected rhesus monkeys lyse autologous B lymphoblastoid cell lines infected with a recombinant vaccinia virus that carries the SIVmac gag gene. This lysis is mediated by CD8+ lymphocytes and is MHC class I restricted. Moreover, these effector lymphocytes do not express the NK cell-associated molecules NKH1 or CD16. These cells are, therefore, CTL. In a limited prospective study of SIVmac-infected rhesus monkeys, the presence of the SIVmac gag-specific CTL activity in PBL correlated with both a reduced efficiency in isolating SIVmac from PBL of these monkeys and their extended survival. This method for assessing SIVmac gag-specific cellular immunity in rhesus monkeys will be important not only in investigating the immunopathogenesis of SIVmac-induced disease, but also in evaluating the capacity of candidate AIDS vaccines to elicit a cell-mediated immune response in this animal model.     

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.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.     

Three epitopic peptides of the simian immunodeficiency virus Nef protein recognized by macaque cytolytic T lymphocytes.

In 8 of 12 experimentally infected macaques, the Nef SIVmac 251 protein was recognized by cytolytic T lymphocytes (CTL) and appeared strongly immunogenic. Here, we report experiments which have been performed by using synthetic peptides to precisely determine the epitopes recognized by macaque CTL. Three epitopes of the Nef protein have been defined as CTL targets in three macaques. The epitopic peptides are located in the central region of the protein, and all of them show high homology with peptides of the human immunodeficiency virus type 1 Nef protein recognized by human CTL in association with several human leukocyte antigen molecules. These results suggest that (i) the Nef protein is a good candidate for vaccination not only because of its early expression but also because of its high immunogenicity for CTL, (ii) long peptides covering the central region of this protein could be used as vaccines and could cross the major histocompatibility complex barrier in a large variety of individuals, and (iii) the rhesus macaque is a good animal model in which to test for protection by CTL.     

Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sj?gren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.     

Elicitation of simian immunodeficiency virus-specific cytotoxic T lymphocytes in mucosal compartments of rhesus monkeys by systemic vaccination

Since most human immunodeficiency virus (HIV) infections are initiated following mucosal exposure to the virus, the anatomic containment or abortion of an HIV infection is likely to require vaccine-elicited cellular immune responses in those mucosal sites. Studying vaccine-elicited mucosal immune responses has been problematic because of the difficulties associated with sampling T lymphocytes from those anatomic compartments. In the present study, we demonstrate that mucosal cytotoxic T lymphocytes (CTL) specific for simian immunodeficiency virus (SIV) and simian HIV can be reproducibly sampled from intestinal mucosal tissue of rhesus monkeys obtained under endoscopic guidance. These lymphocytes recognize peptide-major histocompatibility complex class I complexes and express gamma interferon on exposure to peptide antigen. Interestingly, systemic immunization of monkeys with plasmid DNA immunogens followed by live recombinant attenuated poxviruses or adenoviruses with genes deleted elicits high-frequency SIV-specific CTL responses in these mucosal tissues. These studies therefore suggest that systemic delivery of potent HIV immunogens may suffice to elicit substantial mucosal CTL responses.     

Analysis of pigtail macaque major histocompatibility complex class I molecules presenting immunodominant simian immunodeficiency virus epitopes

Successful human immunodeficiency virus (HIV) vaccines will need to induce effective T-cell immunity. We studied immunodominant simian immunodeficiency virus (SIV) Gag-specific T-cell responses and their restricting major histocompatibility complex (MHC) class I alleles in pigtail macaques (Macaca nemestrina), an increasingly common primate model for the study of HIV infection of humans. CD8+ T-cell responses to an SIV epitope, Gag164-172KP9, were present in at least 15 of 36 outbred pigtail macaques. The immunodominant KP9-specific response accounted for the majority (mean, 63%) of the SIV Gag response. Sequencing from six macaques identified 7 new Mane-A and 13 new Mane-B MHC class I alleles. One new allele, Mane-A*10, was common to four macaques that responded to the KP9 epitope. We adapted reference strand-mediated conformational analysis (RSCA) to MHC class I genotype M. nemestrina. Mane-A*10 was detected in macaques presenting KP9 studied by RSCA but was absent from non-KP9-presenting macaques. Expressed on class I-deficient cells, Mane-A*10, but not other pigtail macaque MHC class I molecules, efficiently presented KP9 to responder T cells, confirming that Mane-A*10 restricts the KP9 epitope. Importantly, naive pigtail macaques infected with SIVmac251 that respond to KP9 had significantly reduced plasma SIV viral levels (log10 0.87 copies/ml; P=0.025) compared to those of macaques not responding to KP9. The identification of this common M. nemestrina MHC class I allele restricting a functionally important immunodominant SIV Gag epitope establishes a basis for studying CD8+ T-cell responses against AIDS in an important, widely available nonhuman primate species.