Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication

The regulatory proteins of human immunodeficiency virus may represent important vaccine targets. Here we assessed the role of Tat-specific cytotoxic T lymphocytes (CTL) in controlling pathogenic simian immunodeficiency virus SIVmac239 replication after using a DNA-prime, vaccinia virus Ankara-boost vaccine regimen. Despite the induction of Tat-specific CTL, there was no significant reduction in either peak or viral set point compared to that of controls.     

Dynamics of simian immunodeficiency virus SIVmac239 infection in pigtail macaques

Pigtail macaques (PTM) are an excellent model for HIV research; however, the dynamics of simian immunodeficiency virus (SIV) SIVmac239 infection in PTM have not been fully evaluated. We studied nine PTM prior to infection, during acute and chronic SIVmac239 infections, until progression to AIDS. We found PTM manifest clinical AIDS more rapidly than rhesus macaques (RM), as AIDS-defining events occurred at an average of 42.17 weeks after infection in PTM compared to 69.56 weeks in RM (P = 0.0018). However, increased SIV progression was not associated with increased viremia, as both peak and set-point plasma viremias were similar between PTM and RM (P = 0.7953 and P = 0.1006, respectively). Moreover, this increased disease progression was not associated with rapid CD4(+) T cell depletion, as CD4(+) T cell decline resembled other SIV/human immunodeficiency virus (HIV) models. Since immune activation is the best predictor of disease progression during HIV infection, we analyzed immune activation by turnover of T cells by BrdU decay and Ki67 expression. We found increased levels of turnover prior to SIV infection of PTM compared to that observed with RM, which may contribute to their increased disease progression rate. These data evaluate the kinetics of SIVmac239-induced disease progression and highlight PTM as a model for HIV infection and the importance of immune activation in SIV disease progression.     

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.     

Gamma interferon-mediated inflammation is associated with lack of protection from intravaginal simian immunodeficiency virus SIVmac239 challenge in simian-human immunodeficiency virus 89.6-immunized rhesus macaques

Although gamma interferon (IFN-gamma) is a key mediator of antiviral defenses, it is also a mediator of inflammation. As inflammation can drive lentiviral replication, we sought to determine the relationship between IFN-gamma-related host immune responses and challenge virus replication in lymphoid tissues of simian-human immunodeficiency virus 89.6 (SHIV89.6)-vaccinated and unvaccinated rhesus macaques 6 months after challenge with simian immunodeficiency virus SIVmac239. Vaccinated-protected monkeys had low tissue viral RNA (vRNA) levels, vaccinated-unprotected animals had moderate tissue vRNA levels, and unvaccinated animals had high tissue vRNA levels. The long-term challenge outcome in vaccinated monkeys was correlated with the relative balance between SIV-specific IFN-gamma T-cell responses and nonspecific IFN-gamma-driven inflammation. Vaccinated-protected monkeys had slightly increased tissue IFN-gamma mRNA levels and a high frequency of IFN-gamma-secreting T cells responding to in vitro SIVgag peptide stimulation; thus, it is likely that they could develop effective anti-SIV cytotoxic T lymphocytes in vivo. In contrast, both high tissue IFN-gamma mRNA levels and strong in vitro SIV-specific IFN-gamma T-cell responses were detected in lymphoid tissues of vaccinated-unprotected monkeys. Unvaccinated monkeys had increased tissue IFN-gamma mRNA levels but weak in vitro anti-SIV IFN-gamma T-cell responses. In addition, in lymphoid tissues of vaccinated-unprotected and unvaccinated monkeys, the increased IFN-gamma mRNA levels were associated with increased Mig/CXCL9, IP-10/CXCL10, and CXCR3 mRNA levels, suggesting that increased Mig/CXCL9 and IP-10/CXCL10 expression resulted in recruitment of CXCR3(+) activated T cells. Thus, IFN-gamma-driven inflammation promotes SIV replication in vaccinated-unprotected and unvaccinated monkeys. Unlike all unvaccinated monkeys, most monkeys vaccinated with SHIV89.6 did not develop IFN-gamma-driven inflammation, but they did develop effective antiviral CD8(+)-T-cell responses.     

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.     

Live attenuated lentivirus infection elicits polyfunctional simian immunodeficiency virus Gag-specific CD8+ T cells with reduced apoptotic susceptibility in rhesus macaques that control virus replication after challenge with pathogenic SIVmac239

HIV-specific CD8+ T cells that secrete multiple cytokines in response to Ag stimulation are associated with the control of virus replication during chronic HIV infection. To determine whether the presence of polyfunctional CD8+ T cell responses distinguishes protected and unprotected monkeys in a live attenuated lentivirus model, SIV Gag peptide-specific CD8+ T cell responses of simian HIV (SHIV) 89.6-vaccinated, SIVmac239-challenged rhesus macaques were compared in two monkeys that controlled challenge virus replication and two that did not. The ratio of Bcl-2+ Gag-specific CD8+ T cells to caspase-3+ Gag-specific CD8+ T cells was higher in the vaccinated-protected animals compared with unprotected monkeys. In addition, polyfunctional SIV-specific CD8+ T cells were consistently detected through 12 wk postchallenge in the protected animals but not in the unprotected animals. In the unprotected monkeys, there was an increased frequency of CD8+ T cells expressing markers associated with effector memory T cells. Further, there was increased annexin V expression in central memory T cells of the unprotected animals before challenge. Thus, monkeys that control viral replication after live attenuated SHIV infection have polyfunctional SIV-specific CD8+ T cells with an increased survival potential. Importantly, the differences in the nature of the SIV-specific CD8+ T cell response in the protected and unprotected animals are present during acute stages postchallenge, before different antigenic levels are established. Thus, the polyfunctional capacity and increased survival potential of CD8+ SIV-specific T cells may account for live attenuated, SHIV89.6-mediated protection from uncontrolled SIV replication.     

Impact of vaccination on cytotoxic T lymphocyte immunodominance and cooperation against simian immunodeficiency virus replication in rhesus macaques

Cytotoxic T lymphocyte (CTL) responses play a central role in viral suppression in human immunodeficiency virus (HIV) infections. Prophylactic vaccination resulting in effective CTL responses after viral exposure would contribute to HIV control. It is important to know how CTL memory induction by vaccination affects postexposure CTL responses. We previously showed vaccine-based control of a simian immunodeficiency virus (SIV) challenge in a group of Burmese rhesus macaques sharing a major histocompatibility complex class I haplotype. Gag(206-216) and Gag(241-249) epitope-specific CTL responses were responsible for this control. In the present study, we show the impact of individual epitope-specific CTL induction by prophylactic vaccination on postexposure CTL responses. In the acute phase after SIV challenge, dominant Gag(206-216)-specific CTL responses with delayed, naive-derived Gag(241-249)-specific CTL induction were observed in Gag(206-216) epitope-vaccinated animals with prophylactic induction of single Gag(206-216) epitope-specific CTL memory, and vice versa in Gag(241-249) epitope-vaccinated animals with single Gag(241-249) epitope-specific CTL induction. Animals with Gag(206-216)-specific CTL induction by vaccination selected for a Gag(206-216)-specific CTL escape mutation by week 5 and showed significantly less decline of plasma viral loads from week 3 to week 5 than in Gag(241-249) epitope-vaccinated animals without escape mutations. Our results present evidence indicating significant influence of prophylactic vaccination on postexposure CTL immunodominance and cooperation of vaccine antigen-specific and non-vaccine antigen-specific CTL responses, which affects virus control. These findings provide great insights into antigen design for CTL-inducing AIDS vaccines.     

Retroviral recombination in vivo: viral replication patterns and genetic structure of simian immunodeficiency virus (SIV) populations in rhesus macaques after simultaneous or sequential intravaginal inoculation with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef

To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Deltavpx/Deltavpr and SIVmac239Deltanef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Deltavpx/Deltavpr and then with SIVmac239Deltanef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Deltanef when plasma SIVmac239Deltavpx/Deltavpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Deltanef, and then with SIVmac239Deltavpx/Deltavpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.     

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.     

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.     

Cytotoxic T-lymphocyte escape does not always explain the transient control of simian immunodeficiency virus SIVmac239 viremia in adenovirus-boosted and DNA-primed Mamu-A*01-positive rhesus macaques

Adenovirus 5 (Ad5) vectors show promise as human immunodeficiency virus vaccine candidates. Indian rhesus macaques vaccinated with Ad5-gag controlled simian-human immunodeficiency virus SHIV89.6P viral replication in the absence of Env immunogens that might elicit humoral immunity. Here we immunized 15 macaques using either a homologous Ad5-gag/Ad5-gag (Ad5/Ad5) or a heterologous DNA-gag/Ad5-gag (DNA/Ad5) prime-boost regimen and challenged them with a high dose of simian immunodeficiency virus SIVmac239. Macaques vaccinated with the DNA/Ad5 regimen experienced a brief viral load nadir of less than 10,000 viral copies per ml blood plasma that was not seen in Mamu-A*01-negative DNA/Ad5 vaccinees, Mamu-A*01-positive Ad5/Ad5 vaccinees, or vaccine-naive controls. Interestingly, most of these animals were not durably protected from disease progression when challenged with SIVmac239. To investigate the reasons underlying this short-lived vaccine effect, we investigated breadth of the T-cell response, immunogenetic background, and viral escape from CD8+ lymphocytes that recognize immunodominant T-cell epitopes. We show that these animals do not mount unusually broad cellular immune response, nor do they express unusual major histocompatibility complex class I alleles. Viral recrudescence occurred in four of the five Mamu-A*01-positive vaccinated macaques. However, only a single animal in this group demonstrated viral escape in the immunodominant Gag181-189 CM9 response. These results suggest that viral "breakthrough" in vaccinated animals and viral escape are not inextricably linked and underscore the need for additional research into the mechanisms of vaccine failure.     

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.     

Correlation between env V1/V2 region diversification and neutralizing antibodies during primary infection by simian immunodeficiency virus sm in rhesus macaques

Evolution of the domain encoding the V1/V2 variable region of the simian immunodeficiency virus sm (SIVsm) envelope (env) gene was analyzed in relation to route of virus challenge, virus load, and neutralizing antibody (NAb) titers during primary infection of rhesus macaques with the pathogenic SIVsmE660 isolate. In this model system animals are initially infected with multiple viruses as evidenced by the presence of multiple V1/V2 genotypic variants that could be resolved by using a heteroduplex tracking assay (HTA). Overlapping subsets of the multiple variants were established in each animal. There was no selection for the establishment of specific variants in comparing intravenous- and intrarectal-challenged macaques at week 2 postinfection, suggesting that no genotypic selection occurred at the mucosal surface. There was an initial period of significant stability of the V1/V2 variants. Macaques challenged intravenously displayed subsequent V1/V2 diversification significantly earlier than macaques challenged intrarectally and well past the initial resolution of viremia. The time when SIVsmE660-specific NAbs reached a threshold titer of 100 was significantly correlated with the timing of V1/V2 diversification, even though antibodies to the Env protein could be detected much earlier. The time when NAbs reached a titer of 400 was significantly correlated with virus load late in infection. These results show that the route of infection affects the timing of V1/V2 diversification and that this diversification is correlated with the maturation of a specific NAb response. However, prior immunization capable of priming an anamnestic Env antibody response did not accelerate V1/V2 diversification. This result suggests that diversification of the SIV env V1/V2 region is the result of a type-specific antibody response.     

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.     

Antibody-dependent cell-mediated viral inhibition emerges after simian immunodeficiency virus SIVmac251 infection of rhesus monkeys coincident with gp140-binding antibodies and is effective against neutralization-resistant viruses

Antibody-dependent cell-mediated viral inhibition (ADCVI) is an attractive target for vaccination because it takes advantage of both the anamnestic properties of an adaptive immune response and the rapid early response characteristics of an innate immune response. Effective utilization of ADCVI in vaccine strategies will depend on an understanding of the natural history of ADCVI during acute and chronic human immunodeficiency virus type 1 (HIV-1) infection. We used the simian immunodeficiency virus (SIV)-infected rhesus monkey as a model to study the kinetics of ADCVI in early infection, the durability of ADCVI through the course of infection, and the effectiveness of ADCVI against viruses with envelope mutations that are known to confer escape from antibody neutralization. We demonstrate the development of ADCVI, capable of inhibiting viral replication 100-fold, within 3 weeks of infection, preceding the development of a comparable-titer neutralizing antibody response by weeks to months. The emergence of ADCVI was temporally associated with the emergence of gp140-binding antibodies, and in most animals, ADCVI persisted through the course of infection. Highly evolved viral envelopes from viruses isolated at late time points following infection that were resistant to plasma neutralization remained susceptible to ADCVI, suggesting that the epitope determinants of neutralization escape are not shared by antibodies that mediate ADCVI. These findings suggest that despite the ability of SIV to mutate and adapt to multiple immunologic pressures during the course of infection, SIV envelope may not escape the binding of autologous antibodies that mediate ADCVI.     

Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus

We studied the innate and adaptive immune system of rhesus macaques infected with the virulent simian immunodeficiency virus isolate SIVmac251 by evaluating natural killer (NK) cell activity, cytokine levels in plasma, humoral and virological parameters, and changes in the activation markers CD25 (interleukin 2R [IL-2R] α chain), CD69 (early activation marker), and CD154 (CD40 ligand) in lymphoid cells. We found that infection with SIVmac251 induced the sequential production of interferon-α/β (IFN-α/β), IL-18, and IL-12. IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor were undetected in plasma by the assays used. NK cell activity peaked at 1 to 2 weeks postinfection and paralleled changes in viral loads. Maximum expression of CD69 on CD3⁻CD16⁺ lymphocytes correlated with NK cytotoxicity during this period. CD25 expression, which is associated with proliferation, was static or slightly down-regulated in CD4⁺ T cells from both peripheral blood (PB) and lymph nodes (LN). CD69, which is normally present in LN CD4⁺ T cells and absent in peripheral blood leukocyte (PBL) CD4⁺ T cells, was down-regulated in LN CD4⁺ T cells and up-regulated in PBL CD4⁺ T cells immediately after infection. CD8⁺ T cells increased CD69 but not CD25 expression, indicating the activation of this cellular subset in PB and LN. Finally, CD154 was transiently up-regulated in PBL CD4⁺ T cells but not in LN CD4⁺ T cells. Levels of antibodies to SIV Gag and Env did not correlate with the level of activation of CD154, a critical costimulatory molecule for T-cell-dependent immunity. In summary, we present the first documented evidence that the innate immune system of rhesus macaques recognizes SIV infection by sequential production of proinflammatory cytokines and transient activation of NK cytotoxic activity. Additionally, pathogenic SIV induces drastic changes in the level of activation markers on T cells from different anatomic compartments. These changes involve activation in the absence of proliferation, indicating that activation-induced cell death may cause some of the reported increase in lymphocyte turnover during SIV infection.The immune system of higher vertebrates consists of innate and adaptive components. Innate immunity exhibits immediate recognition and response without prior sensitization. Cells of the innate immune system (i.e., monocytes/macrophages, natural killer [NK] cells, and polymorphonuclear leukocytes) recognize pathogen-associated molecular patterns and activate events such as phagocytosis, induction of the synthesis of antimicrobial peptides, expression of inflammatory and effector cytokines and chemokines, induction of nitric oxide synthase in macrophages, and expression of costimulatory molecules on antigen-presenting cells. The adaptive immune system uses somatically generated antigen receptors that are clonally distributed on T and B lymphocytes. Generally, adaptive immune recognition in the absence of innate immune recognition results in inactivation of lymphocytes that express receptors involved in the identification events (20). Thus, innate immune responses have critical consequences in adaptive immune responses.Little is known of the contribution of the innate immune system during infection with the human immunodeficiency virus (HIV). Based on similarities of biologic and genetic features, simian immunodeficiency virus (SIV) infection of rhesus macaques provides the best animal model of HIV infection and AIDS. Accordingly, this animal model is critical for the elucidation of mechanisms of pathogenesis and for the development of vaccines and antiviral therapies (12). As with almost all viral infections, the innate immune system is thought to be the first component of the immune system that recognizes SIV infection. However, few studies have methodically analyzed the changes induced in cell phenotype and cytokine levels by SIV infection. Recent studies have demonstrated that SIV infection results in a generalized increase in lymphocyte turnover (23) and that the primary site for viral replication is activated memory CD4⁺ T cells that are present in the intestinal lamina propia (46). Although cellular changes are not that dramatic at this early stage in peripheral lymphoid tissue, peripheral blood (PB) and lymph nodes (LN) still reflect the pathologic changes induced by the viral infection and are readily available for longitudinal studies.To analyze changes in the activation state of cells from the innate and adaptive immune system after SIV infection, we evaluated NK activity, cytokine levels in plasma, and changes in activation markers on lymphoid cells of rhesus macaques after infection with pathogenic SIVmac251. We found the sequential appearance in plasma of interferon-α/β (IFN-α/β) interleukin-18 (IL-18) and IL-12, whereas IL-4, IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) remained undetectable. We also found transient activation of NK cells during the peak of viral replication, and this activation was not predictive of disease progression. Finally, we observed that after SIV infection, both CD4⁺ and CD8⁺ T cells became activated in the absence of markers for proliferation, suggesting that the increased turnover of these cells reflects activation-induced cell death rather than differential compartmentalization.     

CD8+ T cell dynamics during primary simian immunodeficiency virus infection in macaques: relationship of effector cell differentiation with the extent of viral replication

Immunological and virological events that occur during the earliest stages of HIV-1 infection are now considered to have a major impact on subsequent disease progression. We observed changes in the frequencies of CD8(bright) T cells expressing different chemokine receptors in the peripheral blood and lymph nodes of rhesus macaques during the acute phase of the pathogenic SIVmac251 infection; the frequency of CD8(bright) T cells expressing CXCR4 decreased, while the frequency of those expressing CCR5 increased. These reciprocal changes in chemokine receptor expression were associated with changes in the proportion of cycling (Ki67(+)) CD8(bright) T cells, and with the pattern of CD8(bright) T cell differentiation as defined by expression of CCR7 and CD45RA. In contrast, during the primary phase of the attenuated SIVmac251Deltanef infection, no major change was observed. Whereas during the acute phase of the infection with pathogenic SIV (2 wk postinfection) no correlate of disease protection was identified, once the viral load set points were established (2 mo postinfection), we found that the levels of cycling and of CCR5- and CXCR4-positive CD8(bright) T cells were correlated with the extent of viral replication and therefore with SIV-infection outcome. Our data reveal that, during primary SIV infection, despite intense CD8 T cell activation and an increase in CCR5 expression, which are considered as essential for optimal effector function of CD8(+) T cells, these changes are associated with a poor prognosis for disease progression to AIDS.     

Immunization with a live, attenuated simian immunodeficiency virus (SIV) prevents early disease but not infection in rhesus macaques challenged with pathogenic SIV.

An infectious, virulence-attenuated molecular clone of simian immunodeficiency virus (SIV), SIVMAC-1A11, was derived from an SIV isolate that causes fatal immunodeficiency in rhesus macaques. When inoculated intravenously in rhesus macaques, SIVMAC-1A11 induced transient viremia (1 to 6 weeks) without clinical disease and a persistent humoral antibody response. The antibodies were directed mainly against the viral envelope glycoproteins, as determined by immunoblots and virus neutralization. The potential of this virulence-attenuated virus to protect against intravenous challenge with a pathogenic SIVMAC strain was assessed. Five rhesus macaques were each given two intravenous inoculations with SIVMAC-1A11 7 months apart. Three of the five immunized monkeys and four naive control animals were then challenged with 100 to 1,000 100% animal infectious doses of pathogenic SIVMAC. All seven animals became persistently viremic following the challenge. Four of four unimmunized animals developed severe clinical signs of simian acquired immunodeficiency syndrome by 38 to 227 days after challenge and were euthanatized 91 to 260 days postchallenge. However, no signs of illness were seen in immunized monkeys until 267 to 304 days postchallenge, when two of three immunized animals developed mild thrombocytopenia and lymphopenia; one of these animals died with clinical signs of simian immunodeficiency disease at 445 days after challenge. The two SIVMAC-1A11-immunized monkeys that were not challenged were healthy and antibody positive 22 months after the initial immunization. Thus, although live SIVMAC-1A11 was immunogenic and did not induce any disease, it failed to protect rhesus macaques against infection with a moderately high dose of pathogenic virus. However, immunization prevented severe, early disease and prolonged the lives of monkeys subsequently infected with pathogenic SIV.     

The relationship between simian immunodeficiency virus RNA levels and the mRNA levels of alpha/beta interferons (IFN-alpha/beta) and IFN-alpha/beta-inducible Mx in lymphoid tissues of rhesus macaques during acute and chronic infection

To define the role of alpha/beta interferons (IFN-alpha/beta) in simian immunodeficiency virus (SIV) infection, IFN-alpha and IFN-beta mRNA levels and mRNA levels of Mx, an antiviral effector molecule, were determined in lymphoid tissues of rhesus macaques infected with pathogenic SIV. IFN-alpha/beta responses were induced during the acute phase and persisted in various lymphoid tissues throughout the chronic phase of infection. IFN-alpha/beta responses were most consistent in tissues with high viral RNA levels; thus, IFN-alpha/beta responses were not generally associated with effective control of SIV replication. IFN-alpha/beta responses were differentially regulated in different lymphoid tissues and at different stages of infection. The most consistent IFN-alpha/beta responses in acute and chronic SIV infection were observed in peripheral lymph nodes. In the spleen, only a transient increase in IFN-alpha/beta mRNA levels during acute SIV infection was observed. Further, IFN-alpha and IFN-beta mRNA levels showed a tissue-specific expression pattern during the chronic, but not the acute, phase of infection. In the acute phase of infection, SIV RNA levels in lymphoid tissues of rhesus macaques correlated with mRNA levels of both IFN-alpha and IFN-beta, whereas during chronic SIV infection only increased IFN-alpha mRNA levels correlated with the level of virus replication in the same tissues. In lymphoid tissues of all SIV-infected monkeys, higher viral RNA levels were associated with increased Mx mRNA levels. We found no evidence that monkeys with increased Mx mRNA levels in lymphoid tissues had enhanced control of virus replication. In fact, Mx mRNA levels were associated with high viral RNA levels in lymphoid tissues of chronically infected animals.     

A replication competent adenovirus 5 host range mutant-simian immunodeficiency virus (SIV) recombinant priming/subunit protein boosting vaccine regimen induces broad,persistent SIV-specific cellular immunity to dominant and subdominant epitopes in Mamu-A*01 rhesus macaques

CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8(+) T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-gamma-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8(+) T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8(+) T cells following in vitro stimulation. The SIV-specific CD8(+) T cells elicited were functional and secreted IFN-gamma in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8(+) T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8(+) T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.