Identification and spontaneous immune targeting of an endogenous retrovirus K envelope protein in the Indian rhesus macaque model of human disease.

BACKGROUND: Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections that have invaded the germ line of both humans and non-human primates. Most ERVs are functionally crippled by deletions, mutations, and hypermethylation, leading to the view that they are inert genomic fossils. However, some ERVs can produce mRNA transcripts, functional viral proteins, and even non-infectious virus particles during certain developmental and pathological processes. While there have been reports of ERV-specific immunity associated with ERV activity in humans, adaptive immune responses to ERV-encoded gene products remain poorly defined and have not been investigated in the physiologically relevant non-human primate model of human disease. FINDINGS: Here, we identified the rhesus macaque equivalent of the biologically active human ERV-K (HML-2), simian ERV-K (SERV-K1), which retains intact open reading frames for both Gag and Env on chromosome 12 in the macaque genome. From macaque cells we isolated a spliced mRNA product encoding SERV-K1 Env, which possesses all the structural features of a canonical, functional retroviral Envelope protein. Furthermore, we identified rare, but robust T cell responses as well as frequent antibody responses targeting SERV-K1 Env in rhesus macaques. CONCLUSIONS: These data demonstrate that SERV-K1 retains biological activity sufficient to induce cellular and humoral immune responses in rhesus macaques. As ERV-K is the youngest and most active ERV family in the human genome, the identification and characterization of the simian orthologue in rhesus macaques provides a highly relevant animal model in which to study the role of ERV-K in developmental and disease states.

Tertiary mutations stabilize CD8+ T lymphocyte escape-associated compensatory mutations following transmission of simian immunodeficiency virus.

Compensatory mutations offset fitness defects resulting from CD8(+) T lymphocyte (CD8(TL))-mediated escape, but their impact on viral evolution following transmission to naive hosts remains unclear. Here, we investigated the reversion kinetics of Gag(181-189)CM9 CD8(TL) escape-associated compensatory mutations in simian immunodeficiency virus (SIV)-infected macaques. Preexisting compensatory mutations did not result in acute-phase escape of the SIVmac239 CD8(TL) epitope Gag(181-189)CM9 and instead required a tertiary mutation for stabilization in the absence of Gag(181-189)CM9 escape mutations. Therefore, transmitted compensatory mutations do not necessarily predict rapid CD8(TL) escape.

Neutralizing polyclonal IgG present during acute infection prevents rapid disease onset in simian-human immunodeficiency virus SHIVSF162P3-infected infant rhesus macaques.

Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression.

CD8+ and CD4+ cytotoxic T cell escape mutations precede breakthrough SIVmac239 viremia in an elite controller.

BACKGROUND: Virus-specific T cells are critical components in the containment of immunodeficiency virus infections. While the protective role of CD8+ T cells is well established by studies of CD8+ T cell-mediated viral escape, it remains unknown if CD4+ T cells can also impose sufficient selective pressure on replicating virus to drive the emergence of high-frequency escape variants. Identifying a high frequency CD4+ T cell driven escape mutation would provide compelling evidence of direct immunological pressure mediated by these cells. RESULTS: Here, we studied a SIVmac239-infected elite controller rhesus macaque with a 1,000-fold spontaneous increase in plasma viral load that preceded disease progression and death from AIDS-related complications. We sequenced the viral genome pre- and post-breakthrough and demonstrate that CD8+ T cells drove the majority of the amino acid substitutions outside of Env. However, within a region of Gag p27CA targeted only by CD4+ T cells, we identified a unique post-breakthrough mutation, Gag D205E, which abrogated CD4+ T cell recognition. Further, we demonstrate that the Gag p27CA-specific CD4+ T cells exhibited cytolytic activity and that SIV bearing the Gag D205E mutation escapes this CD4+ T cell effector function ex vivo. CONCLUSIONS: Cumulatively, these results confirm the importance of virus specific CD8+ T cells and demonstrate that CD4+ T cells can also exert significant selective pressure on immunodeficiency viruses in vivo during low-level viral replication. These results also suggest that further studies of CD4+ T cell escape should focus on cases of elite control with spontaneous viral breakthrough.

Experimental analysis of sources of error in evolutionary studies based on Roche/454 pyrosequencing of viral genomes.

Factors affecting the reliability of Roche/454 pyrosequencing for analyzing sequence polymorphism in within-host viral populations were assessed by two experiments: 1) sequencing four clonal simian immunodeficiency virus (SIV) stocks and 2) sequencing mixtures in different proportions of two SIV strains with known fixed nucleotide differences. Observed nucleotide diversity and frequency of undetermined nucleotides were increased at sites in homopolymer runs of four or more identical nucleotides, particularly at AT sites. However, in the mixed-strain experiments, the effects on estimated nucleotide diversity of such errors were small in comparison to known strain differences. The results suggest that biologically meaningful variants present at a frequency of around 10% and possibly much lower are easily distinguished from artifacts of the sequencing process. Analysis of the clonal stocks revealed numerous rare variants that showed the signature of purifying selection and that elimination of variants at frequencies of less than 1% reduced estimates of nucleotide diversity by about an order of magnitude. Thus, using a 1% frequency cutoff for accepting a variant as real represents a conservative standard, which may be useful in studies that are focused on the discovery of specific mutations (such as those conferring immune escape or drug resistance). On the other hand, if the goal is to estimate nucleotide diversity, an optimal strategy might be to include all observed variants (even those at less than 1% frequency), while masking out homopolymer runs of four or more nucleotides.

CD8+ T cell escape mutations in simian immunodeficiency virus SIVmac239 cause fitness defects in vivo, and many revert after transmission.

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.

GagCM9-specific CD8+ T cells expressing limited public TCR clonotypes do not suppress SIV replication in vivo.

Several lines of evidence suggest that HIV/SIV-specific CD8(+) T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag(45-269)) that were subsequently infected with SIVsmE660. These seven Mamu-A*01(+) animals developed CD8(+) T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8(+) T cells could not control virus replication in vivo. GagCM9-specific CD8(+) T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8(+) T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20-250 GagCM9-specific CD8(+) T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8(+) T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8(+) T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8(+) T cell population elicited by vaccination and infection.

The role of MHC class I allele Mamu-A*07 during SIV(mac)239 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.

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.

Macaques vaccinated with simian immunodeficiency virus SIVmac239Delta nef delay acquisition and control replication after repeated low-dose heterologous SIV challenge.

An effective human immunodeficiency virus (HIV) vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best simian immunodeficiency virus (SIV) vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Delta nef and challenged them intrarectally (i.r.) with repeated low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to that for naïve controls (log rank test; P = 0.023). After 10 mucosal challenges, we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these 10 challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than did the naïve controls (Mann-Whitney U test; P = 0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 postinfection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequence analysis of the circulating viruses in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.

High viremia is associated with high levels of in vivo major histocompatibility complex class I Downregulation in rhesus macaques infected with simian immunodeficiency virus SIVmac239.

Human and simian immunodeficiency viruses (HIV and SIV) downregulate major histocompatibility complex class I (MHC-I) molecules from the surface of infected cells. Although this activity is conserved across viral isolates, its importance in AIDS pathogenesis is not clear. We therefore developed an assay to detect the level of MHC-I expression of SIV-infected cells directly ex vivo. Here we show that the extent of MHC-I downregulation is greatest in SIVmac239-infected macaques that never effectively control virus replication. Our results suggest that a high level of MHC-I downregulation is a hallmark of fast disease progression in SIV infection.

Infection with "escaped" virus variants impairs control of simian immunodeficiency virus SIVmac239 replication in Mamu-B*08-positive macaques.

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8+ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8+ T-cell epitopes to investigate the contribution of epitope-specific CD8+ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08+ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08+ macaques. The five most immunodominant Mamu-B*08-restricted CD8+ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08+ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08+ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8+ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08+ macaques.

Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection.

The precise immunological role played by CD4(+) T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8(+) cell depletion, elite controlling macaques with set-point viral loads < or = 500 viral RNA copies/mL mounted robust Gag- and Nef-specific CD4(+) T cell responses during reestablishment of control with > or = 54% of all virus-specific CD4(+) T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4(+) T cells neither recognized nor suppressed viral replication in SIV-infected CD4(+) T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4(+) T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4(+) T cells may contribute directly to elite control by inhibiting viral replication in macrophages.

Macaques vaccinated with live-attenuated SIV control replication of heterologous virus.

An effective AIDS vaccine will need to protect against globally diverse isolates of HIV. To address this issue in macaques, we administered a live-attenuated simian immunodeficiency virus (SIV) vaccine and challenged with a highly pathogenic heterologous isolate. Vaccinees reduced viral replication by approximately 2 logs between weeks 2-32 (P < or = 0.049) postchallenge. Remarkably, vaccinees expressing MHC-I (MHC class I) alleles previously associated with viral control completely suppressed acute phase replication of the challenge virus, implicating CD8(+) T cells in this control. Furthermore, transient depletion of peripheral CD8(+) lymphocytes in four vaccinees during the chronic phase resulted in an increase in virus replication. In two of these animals, the recrudescent virus population contained only the vaccine strain and not the challenge virus. Alarmingly, however, we found evidence of recombinant viruses emerging in some of the vaccinated animals. This finding argues strongly against an attenuated virus vaccine as a solution to the AIDS epidemic. On a more positive note, our results suggest that MHC-I-restricted CD8(+) T cells contribute to the protection induced by the live-attenuated SIV vaccine and demonstrate that vaccine-induced CD8(+) T cell responses can control replication of heterologous challenge viruses.

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.

Patterns of CD8+ immunodominance may influence the ability of Mamu-B*08-positive macaques to naturally control simian immunodeficiency virus SIVmac239 replication.

Certain major histocompatibility complex (MHC) class I alleles are strongly associated with control of human immunodeficiency virus and simian immunodeficiency virus (SIV). CD8(+) T cells specific for epitopes restricted by these molecules may be particularly effective. Understanding how CD8(+) T cells contribute to control of viral replication should yield important insights for vaccine design. We have recently identified an Indian rhesus macaque MHC class I allele, Mamu-B*08, associated with elite control and low plasma viremia after infection with the pathogenic isolate SIVmac239. Here, we infected four Mamu-B*08-positive macaques with SIVmac239 to investigate why some of these macaques control viral replication. Three of the four macaques controlled SIVmac239 replication with plasma virus concentrations below 20,000 viral RNA copies/ml at 20 weeks postinfection; two of four macaques were elite controllers (ECs). Interestingly, two of the four macaques preserved their CD4(+) memory T lymphocytes during peak viremia, and all four recovered their CD4(+) memory T lymphocytes in the chronic phase of infection. Mamu-B*08-restricted CD8(+) T-cell responses dominated the acute phase and accounted for 23.3% to 59.6% of the total SIV-specific immune responses. Additionally, the ECs mounted strong and broad CD8(+) T-cell responses against several epitopes in Vif and Nef. Mamu-B*08-specific CD8(+) T cells accounted for the majority of mutations in the virus at 18 weeks postinfection. Interestingly, patterns of viral variation in Nef differed between the ECs and the other two macaques. Natural containment of AIDS virus replication in Mamu-B*08-positive macaques may, therefore, be related to a combination of immunodominance and viral escape from CD8(+) T-cell responses.

CD8+ T cells from SIV elite controller macaques recognize Mamu-B*08-bound epitopes and select for widespread viral variation.

BACKGROUND: It is generally accepted that CD8+ T cell responses play an important role in control of immunodeficiency virus replication. The association of HLA-B27 and -B57 with control of viremia supports this conclusion. However, specific correlates of viral control in individuals expressing these alleles have been difficult to define. We recently reported that transient in vivo CD8+ cell depletion in simian immunodeficiency virus (SIV)-infected elite controller (EC) macaques resulted in a brief period of viral recrudescence. SIV replication was rapidly controlled with the reappearance of CD8+ cells, implicating that these cells actively suppress viral replication in ECs. METHODS AND FINDINGS: Here we show that three ECs in that study made at least seven robust CD8+ T cell responses directed against novel epitopes in Vif, Rev, and Nef restricted by the MHC class I molecule Mamu-B*08. Two of these Mamu-B*08-positive animals subsequently lost control of SIV replication. Their breakthrough virus harbored substitutions in multiple Mamu-B*08-restricted epitopes. Indeed, we found evidence for selection pressure mediated by Mamu-B*08-restricted CD8+ T cells in all of the newly identified epitopes in a cohort of chronically infected macaques. CONCLUSIONS: Together, our data suggest that Mamu-B*08-restricted CD8+ T cell responses effectively control replication of pathogenic SIV(mac)239. All seven regions encoding Mamu-B*08-restricted CD8+ T cell epitopes also exhibit amino acid replacements typically seen only in the presence of Mamu-B*08, suggesting that the variation we observe is indeed selected by CD8+ T cell responses. SIV(mac)239 infection of Indian rhesus macaques expressing Mamu-B*08 may therefore provide an animal model for understanding CD8+ T cell-mediated control of HIV replication in humans.

Subdominant CD8+ T-cell responses are involved in durable control of AIDS virus replication.

"Elite controllers" are individuals that durably control human immunodeficiency virus or simian immunodeficiency virus replication without therapeutic intervention. The study of these rare individuals may facilitate the definition of a successful immune response to immunodeficiency viruses. Here we describe six Indian-origin rhesus macaques that have controlled replication of the pathogenic virus SIVmac239 for 1 to 5 years. To determine which lymphocyte populations were responsible for this control, we transiently depleted the animals' CD8+ cells in vivo. This treatment resulted in 100- to 10,000-fold increases in viremia. When the CD8+ cells returned, control was reestablished and the levels of small subsets of previously subdominant CD8+ T cells expanded up to 2,500-fold above pre-depletion levels. This wave of CD8+ T cells was accompanied by robust Gag-specific CD4 responses. In contrast, CD8+ NK cell frequencies changed no more than threefold. Together, our data suggest that CD8+ T cells targeting a small number of epitopes, along with broad CD4+ T-cell responses, can successfully control the replication of the AIDS virus. It is likely that subdominant CD8+ T-cell populations play a key role in maintaining this control.