Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

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.     

Containment of simian immunodeficiency virus infection in vaccinated macaques: correlation with the magnitude of virus-specific pre- and postchallenge CD4+ and CD8+ T cell responses

Macaques infected with the SIV strain SIVmac251 develop a disease closely resembling human AIDS characterized by high viremia, progressive loss of CD4(+) T cells, occurrence of opportunistic infection, cachexia, and lymphomas. We report in this study that vaccination with the genetically attenuated poxvirus vector expressing the structural Ags of SIVmac (NYVAC-SIV-gag, pol, env) in combination with priming with DNA-SIV-gag, env resulted in significant suppression of viremia within 2 mo after mucosal exposure to the highly pathogenic SIVmac251 in the majority of vaccinated macaques. The control of viremia in these macaques was long lasting and inversely correlated to the level of both pre- and postchallenge Gag-specific lymphoproliferative responses, as well as to the level of total SIV-specific CD4(+) T lymphocyte responses at the peak of acute viremia as detected by intracellular cytokine-staining assay. Viremia containment also correlated with the frequency of the immunodominant Gag(181-189)CM9 epitope-specific CD8(+) T cells present before the challenge or expanded during acute infection. These data indicate, for the first time, the importance of vaccine-induced CD4(+) Th cell responses as an immune correlate of viremia containment. The results presented in this work also further demonstrate the potential of a DNA-prime/attenuated poxvirus-boost vaccine regimen in an animal model that well mirrors human AIDS.     

The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.     

Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.     

Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac239delta3-vaccinated rhesus macaques

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.     

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.     

DNA vaccines expressing different forms of simian immunodeficiency virus antigens decrease viremia upon SIVmac251 challenge

We have tested the efficacy of DNA immunization as a single vaccination modality for rhesus macaques followed by highly pathogenic SIVmac251 challenge. To further improve immunogenicity of the native proteins, we generated expression vectors producing fusion of the proteins Gag and Env to the secreted chemokine MCP3, targeting the viral proteins to the secretory pathway and to a beta-catenin (CATE) peptide, targeting the viral proteins to the intracellular degradation pathway. Macaques immunized with vectors expressing the MCP3-tagged fusion proteins developed stronger antibody responses. Following mucosal challenge with pathogenic SIVmac251, the vaccinated animals showed a statistically significant decrease in viral load (P = 0.010). Interestingly, macaques immunized with a combination of vectors expressing three forms of antigens (native protein and MCP3 and CATE fusion proteins) showed the strongest decrease in viral load (P = 0.0059). Postchallenge enzyme-linked immunospot values for Gag and Env as well as gag-specific T-helper responses correlated with control of viremia. Our data show that the combinations of DNA vaccines producing native and modified forms of antigens elicit more balanced immune responses able to significantly reduce viremia for a long period (8 months) following pathogenic challenge with SIVmac251.     

T-cell-mediated protective efficacy of a systemic vaccine approach in cynomolgus monkeys after SIV mucosal challenge

The immunogenicity and the protective efficacy of a new polyvalent triple vector (DNA/SFV/MVA) based vaccine against mucosal challenge with pathogenic SIVmac251 were investigated. Cynomolgus monkeys (Macaca fascicularis) were primed intradermally with DNA, boosted twice subcutaneously with recombinant Semliki Forest virus (rSFV) and finally intramuscularly with recombinant Modified Vaccinia Virus Ankara strain (rMVA). Both DNA and recombinant viral vectors expressed SIV proteins (Gag, Pol, Tat, Rev, Nef and Env). The vaccinated monkeys developed T helper proliferative responses to viral antigens after the second immunization while interferon (IFN)-gamma enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) specific responses appeared only after the last boost with rMVA. Upon intrarectal challenge with pathogenic SIVmac251, three of four vaccinated monkeys were either fully protected or exhibited a dramatic reduction of virus replication up to undetectable level. A major contribution to this protective effect appeared to be the anamnestic T-cell IFN-gamma ELISPOT responses to vaccine antigens (Gag, Rev, Tat, Nef) that mirrored the viral clearance. These results underline the efficacy of a multiprotein approach in combination with a triple vector system of antigen delivery.     

Pathogenic conversion of live attenuated simian immunodeficiency virus vaccines is associated with expression of truncated Nef

Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Delta(152)nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.     

Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIV(mac251) challenge by a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen

In this study we investigated the ability of a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen to induce protective immunity in rhesus macaques against pathogenic simian immunodeficiency virus(mac251). Immunization of macaques by two sequential administrations of the same recombinants by the same route resulted in boosting and persistence of SIV-specific cellular immune responses for 42 weeks past the initial immunization. Anti-SIV gp120 immunoglobulin G (IgG) and IgA antibodies were induced in secretory fluids, and all macaques exhibited serum neutralizing antibody activity. After intrarectal SIV(mac251) challenge, all of the macaques became infected. However, relative protection, as assessed by statistically significant lower SIV viral loads in plasma at both acute infection and set point, was observed in 8 out of 12 immunized non-Mamu-A(*)01 animals. Elevated mean cellular immune responses to Gag and Env, neutralizing antibody activity, and IgG and IgA binding antibody levels were observed in the eight protected macaques. Statistically significant correlations with protective outcome were observed for cellular immune responses to SIV Env and Gag and for SIV gp120-specific IgG antibodies in nasal and vaginal fluids. Two macaques that exhibited the greatest and most persistent viremia control also exhibited strong CD8(+) T-cell antiviral activity. The results suggest that a spectrum of immune responses may be necessary for adequate control of viral replication and disease progression and highlight a potential role for nonneutralizing antibodies at mucosal sites.     

Distinct cycling CD4(+)- and CD8(+)-T-cell profiles during the asymptomatic phase of simian immunodeficiency virus SIVmac251 infection in rhesus macaques

Elevated CD4 T-cell turnover may lead to the exhaustion of the immune system during human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, this hypothesis remains controversial. Most studies of this subject have concerned the blood, and information about the lymph nodes is rare and controversial. We used Ki67 expression to measure cycling T cells in the blood and lymph nodes of uninfected macaques and of macaques infected with a pathogenic SIVmac251 strain or with a nonpathogenic SIVmac251Deltanef clone. During the asymptomatic phase of infection, the number of cycling CD8(+) T cells progressively increased (two- to eightfold) both in the blood and in the lymph nodes of macaques infected with SIVmac251. This increase was correlated with viral replication and the progression to AIDS. In contrast, no increases in the numbers of cycling CD4(+) T cells were found in the blood or lymph nodes of macaques infected with the pathogenic SIVmac251 strain in comparison with SIVmac251Deltanef-infected or healthy macaques during this chronic phase. However, the lymph nodes of pre-AIDS stage SIVmac251-infected macaques contained more cycling CD4(+) T cells (low baseline CD4(+)-T-cell counts in the blood). Taken together, these results show that the profiles of CD4(+)- and CD8(+)-T-cell dynamics are distinct both in the lymph nodes and blood and suggest that higher CD4(+)-T-cell proliferation at the onset of AIDS may lead to the exhaustion of the immune system.     

Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus.

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.     

Increased cellular immune responses and CD4+ T-cell proliferation correlate with reduced plasma viral load in SIV challenged recombinant simian varicella virus - simian immunodeficiency virus (rSVV-SIV) vaccinated rhesus macaques

ABSTRACT: BACKGROUND: An effective AIDS vaccine remains one of the highest priorities in HIV-research. Our recent study showed that vaccination of rhesus macaques with recombinant simian varicella virus (rSVV) vector -- simian immunodeficiency virus (SIV) envelope and gag genes, induced neutralizing antibodies and cellular immune responses to SIV and also significantly reduced plasma viral loads following intravenous pathogenic challenge with SIVMAC251/CX1. FINDINGS: The purpose of this study was to define cellular immunological correlates of protection in rSVV-SIV vaccinated and SIV challenged animals. Immunofluorescent staining and multifunctional assessment of SIV-specific T-cell responses were evaluated in both Experimental and Control vaccinated animal groups. Significant increases in the proliferating CD4+ T-cell population and polyfunctional T-cell responses were observed in all Experimental-vaccinated animals compared with the Control-vaccinated animals. CONCLUSIONS: Increased CD4+ T-cell proliferation was significantly and inversely correlated with plasma viral load. Increased SIV-specific polyfunctional cytokine responses and increased proliferation of CD4+ T-cell may be crucial to control plasma viral loads in vaccinated and SIVMAC251/CX1 challenged macaques.     

Induction of CD8+ cells able to suppress CCR5-tropic simian immunodeficiency virus SIVmac239 replication by controlled infection of CXCR4-tropic simian-human immunodeficiency virus in vaccinated rhesus macaques

Recent recombinant viral vector-based AIDS vaccine trials inducing cellular immune responses have shown control of CXCR4-tropic simian-human immunodeficiency virus (SHIV) replication but difficulty in containment of pathogenic CCR5-tropic simian immunodeficiency virus (SIV) in rhesus macaques. In contrast, controlled infection of live attenuated SIV/SHIV can confer the ability to contain SIV superchallenge in macaques. The specific immune responses responsible for this control may be induced by live virus infection but not consistently by viral vector vaccination, although those responses have not been determined. Here, we have examined in vitro anti-SIV efficacy of CD8+ cells in rhesus macaques that showed prophylactic viral vector vaccine-based control of CXCR4-tropic SHIV89.6PD replication. Analysis of the effect of CD8+ cells obtained at several time points from these macaques on CCR5-tropic SIVmac239 replication in vitro revealed that CD8+ cells in the chronic phase after SHIV challenge suppressed SIV replication more efficiently than those before challenge. SIVmac239 superchallenge of two of these macaques at 3 or 4 years post-SHIV challenge was contained, and the following anti-CD8 antibody administration resulted in transient CD8+ T-cell depletion and appearance of plasma SIVmac239 viremia in both of them. Our results indicate that CD8+ cells acquired the ability to efficiently suppress SIV replication by controlled SHIV infection, suggesting the contribution of CD8+ cell responses induced by controlled live virus infection to containment of HIV/SIV superinfection.     

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.     

Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.     

Simian immunodeficiency virus disease course is predicted by the extent of virus replication during primary infection

To elucidate the relationship between early viral infection events and immunodeficiency virus disease progression, quantitative-competitive and branched-DNA methods of simian immunodeficiency virus (SIV) RNA quantitation were cross-validated and used to measure viremia following infection of rhesus macaques with the pathogenic SIVmac251 virus isolate. Excellent correlation between the methods suggests that both accurately approximate SIV copy number. Plasma viremia was evident 4 days postinfection, and rapid viral expansion led to peak viremia levels of 10(7) to 10(9) SIV RNA copies/ml by days 8 to 17. Limited resolution of primary viremia was accompanied by relatively short, though variable, times to the development of AIDS (81 to 630 days). The persistent high-level viremia observed following intravenous inoculation of SIVmac251 explains the aggressive disease course in this model. Survival analyses demonstrated that the disease course is established 8 to 17 days postinfection, when peak viremia is observed. The most significant predictor of disease progression was the extent of viral decline following peak viremia; larger decrements in viremia were associated with both lower steady-state viremia (P = 0.0005) and a reduced hazard of AIDS (P = 0.004). The data also unexpectedly suggested that following SIVmac251 infection, animals with the highest peak viremia were better able to control virus replication rather than more rapidly developing disease. Analysis of early viral replication dynamics should help define host responses that protect from disease progression and should provide quantitative measures to assess the extent to which protective responses may be induced by prophylactic vaccination.     

High frequency of virus-specific CD8+ T cells in the central nervous system of macaques chronically infected with simian immunodeficiency virus SIVmac251

Infection with human immunodeficiency virus or simian immunodeficiency virus (SIV) induces virus-specific CD8(+) T cells that traffic to lymphoid and nonlymphoid tissues. In this study, we used Gag-specific tetramer staining to investigate the frequency of CD8(+) T cells in peripheral blood and the central nervous system of Mamu-A*01-positive SIV-infected rhesus macaques. Most of these infected macaques were vaccinated prior to SIVmac251 exposure. The frequency of Gag(181-189) CM9 tetramer-positive cells was consistently higher in the cerebrospinal fluid and the brain than in the blood of all animals studied and did not correlate with either plasma viremia or CD4(+)-T-cell level. Little or no infection in the brain was documented for most animals by nucleic acid sequence-based amplification or in situ hybridization. These data suggest that this Gag-specific response may contribute to the containment of viral replication in this locale.     

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.