Early induction of polyfunctional simian immunodeficiency virus (SIV)-specific T lymphocytes and rapid disappearance of SIV from lymph nodes of sooty mangabeys during primary infection

Although the cellular immune response is essential for controlling SIV replication in Asian macaques, its role in maintaining nonpathogenic SIV infection in natural hosts such as sooty mangabeys (SM) remains to be defined. We have previously shown that similar to rhesus macaques (RM), SM are able to mount a T lymphocyte response against SIV infection. To investigate early control of SIV replication in natural hosts, we performed a detailed characterization of SIV-specific cellular immunity and viral control in the first 6 mo following SIV infection in SM. Detection of the initial SIV-specific IFN-γ ELISPOT response in SIVsmE041-infected SM coincided temporally with a decline in peak plasma viremia and was similar in magnitude, specificity, and breadth to SIVsmE041-infected and SIVmac239-infected RM. Despite these similarities, SM showed a greater reduction in postpeak plasma viremia and a more rapid disappearance of productively SIV-infected cells from the lymph node compared with SIVmac239-infected RM. The early Gag-specific CD8(+) T lymphocyte response was significantly more polyfunctional in SM compared with RM, and granzyme B-positive CD8(+) T lymphocytes were present at significantly higher frequencies in SM even prior to SIV infection. These findings suggest that the early SIV-specific T cell response may be an important determinant of lymphoid tissue viral clearance and absence of lymph node immunopathology in natural hosts of SIV infection.     

Diverse Host Responses and Outcomes following Simian Immunodeficiency Virus SIVmac239 Infection in Sooty Mangabeys and Rhesus Macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10⁵ to 10⁷ RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8⁺ T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4⁺ T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4⁺ T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.     

Inhibition of Simian Immunodeficiency Virus (SIV) Replication by CD8+ T Lymphocytes from Macaques Immunized with Live Attenuated SIV

Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8⁺ T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Δnef or SIVmac239Δ3 to inhibit SIV replication. CD8⁺ T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4⁺ T cells. Suppression of SIV replication by unstimulated CD8⁺ T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8⁺ T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8⁺ T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8⁺ cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1α (MIP-1α), or MIP-1β from stimulated CD8⁺ T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8⁺ T cells of control animals. However, addition of antibodies that neutralize these β-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8⁺ T cells. Our results indicate that inhibition of SIV replication by CD8⁺ T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1α, and MIP-1β.     

Identification of multiple simian immunodeficiency virus (SIV)-specific CTL epitopes in sooty mangabeys with natural and experimentally acquired SIV infection

Host immune responses to SIV infection in sooty mangabeys are likely to be an important determinant of how such nonhuman primate species maintain asymptomatic lentivirus infection. We have previously described two patterns of asymptomatic SIV infection in sooty mangabeys: low viral loads with vigorous SIV-specific CTL activity in SIVmac239-infected sooty mangabeys, and high viral loads with generally weak or absent SIV-specific CTL activity in naturally infected sooty mangabeys. To define the specificity of the CTL response in SIV-infected mangabeys, we characterized CTL epitopes in two naturally infected and three SIVmac239-infected sooty mangabeys. Compared with that in SIVmac239-infected mangabeys, the yield of SIV-specific CTL clones was significantly lower in naturally infected sooty mangabeys. All CTL clones were phenotypically CD3+ CD8+, and lysis was MHC restricted. Seven SIV CTL epitopes were identified in five sooty mangabeys: one in Gag and three each in Nef and Envelope (Env). The CTL epitopes mapped to conserved regions in the SIV genome and were immunodominant. Several similar or identical CTL epitopes were recognized by both naturally infected and SIVmac239-infected mangabeys that shared class I MHC alleles. To our knowledge, this is the first report of SIV-specific CTL epitopes in sooty mangabeys. Longitudinal studies of viral load and sequence variation in CTL epitopes may provide useful information on the role of CTL in control or persistence of SIV infection in sooty mangabeys.     

Th-1-type cytotoxic CD8+ T-lymphocyte responses to simian immunodeficiency virus (SIV) are a consistent feature of natural SIV infection in sooty mangabeys

Sooty mangabeys are a natural host of simian immunodeficiency virus (SIV) that remain asymptomatic and do not exhibit increased immune activation or increased T-lymphocyte turnover despite sustained high levels of SIV viremia. In this study we asked whether an altered immune response to SIV contributes to the lack of immunopathology in sooty mangabeys as opposed to species with pathogenic lentivirus infection. SIV-specific cellular immune responses were investigated in a cohort of 25 sooty mangabeys with natural SIV infection. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses targeting a median of four SIV proteins were detected in all 25 mangabeys and were comparable in magnitude to those of 13 rhesus macaques infected with SIVmac251 for more than 6 months. As with rhesus macaques, Th2 ELISPOT responses to SIV were absent or >10-fold lower than the IFN-gamma ELISPOT response to the same SIV protein. The SIV-specific ELISPOT response was predominantly mediated by CD8+ T lymphocytes; the frequency of circulating SIV-specific CD8+ T lymphocytes ranged between 0.11% and 3.26% in 13 mangabeys. Functionally, the SIV-specific CD8+ T lymphocytes were cytotoxic; secreted IFN-gamma, tumor necrosis factor alpha, and macrophage inflammatory protein 1beta; and had an activated effector phenotype. Although there was a trend toward higher frequencies of SIV-specific CD8+ T lymphocytes in mangabeys with lower viral loads, a significant inverse correlation between SIV viremia and SIV-specific cellular immunity was not detected. The consistent detection of Th1-type SIV-specific cellular immune responses in naturally infected sooty mangabeys suggests that immune attenuation is neither a feature of nor a requirement for maintenance of nonpathogenic SIV infection in its natural host.     

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4⁺ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4⁺ CCR5⁺ T cells, as this subset of memory/activated CD4⁺ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4⁺ CCR5⁺ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4⁺ CCR5⁺ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4⁺ T-cell function, as measured by CD4⁺ T-cell count, the fraction of memory CD4⁺ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4⁺ CCR5⁺ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4⁺ CCR5⁺ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4⁺ CCR5⁺ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4⁺ CCR5⁺ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.     

Intestinal Intraepithelial Lymphocytes Are Primed for Gamma Interferon and MIP-1β Expression and Display Antiviral Cytotoxic Activity despite Severe CD4+ T-Cell Depletion in Primary Simian Immunodeficiency Virus Infection

Intraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-γ] and MIP-1β) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4⁺ single-positive and CD4⁺CD8⁺ double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8⁺ T cells. This was in contrast to a gradual depletion of CD4⁺ T cells in peripheral blood. The CD8⁺ IEL were the primary producers of IFN-γ and MIP-1β and were found to retain their potential to produce both IFN-γ and MIP-1β through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4⁺ T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.     

Inactivated Simian Immunodeficiency Virus-Pulsed Autologous Fresh Blood Cells as an Immunotherapy Strategy

Practical immunotherapies for human immunodeficiency virus infection are needed. We evaluated inactivated simian immunodeficiency virus (SIV) pulsed onto fresh peripheral blood mononuclear cells in 12 pigtail macaques with chronic SIV_mac251 infection for T-cell immunogenicity in a randomized cross-over design study. The immunotherapy was safe and convincingly induced high levels of SIV-specific CD4⁺ T-cell responses (mean, 5.9% ± 1.3% of all CD4⁺ T cells) and to a lesser extent SIV-specific CD8⁺ T-cell responses (mean, 0.7% ± 0.4%). Responses were primarily directed toward Gag and less frequently toward Env but not Pol or regulatory/accessory SIV proteins. T-cell responses against Gag were generally broad and polyfunctional, with a mean of 2.7 CD4⁺ T-cell epitopes mapped per animal and more than half of the SIV Gag-specific CD4⁺ T cells expressing three or more effector molecules. The immunogenicity was comparable to that found in previous studies of peptide-pulsed blood cells. Despite the high-level immunogenicity, no reduction in viral load was observed in the chronically viremic macaques. This contrasts with our studies of immunization with peptide-pulsed blood cells during early SIV infection in macaques. Future studies of inactivated virus-pulsed blood cell immunotherapy during early infection of patients receiving antiretroviral therapy are warranted.     

Extralymphoid CD8+ T Cells Resident in Tissue from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Suppress SIVmac239 Replication Ex Vivo

Live-attenuated vaccination with simian immunodeficiency virus (SIV) SIVmac239Δnef is the most successful vaccine product tested to date in macaques. However, the mechanisms that explain the efficacy of this vaccine remain largely unknown. We utilized an ex vivo viral suppression assay to assess the quality of the immune response in SIVmac239Δnef-immunized animals. Using major histocompatibility complex-matched Mauritian cynomolgus macaques, we did not detect SIV-specific functional immune responses in the blood by gamma interferon (IFN-γ) enzyme-linked immunospot assay at select time points; however, we found that lung CD8⁺ T cells, unlike blood CD8⁺ T cells, effectively suppress virus replication by up to 80%. These results suggest that SIVmac239Δnef may be an effective vaccine because it elicits functional immunity at mucosal sites. Moreover, these results underscore the limitations of relying on immunological measurements from peripheral blood lymphocytes in studies of protective immunity to HIV/SIV.Despite over 25 years of intensive research, efforts to develop a successful prophylactic HIV vaccine have failed (6, 39). The extraordinary difficulty of developing an HIV vaccine underscores the fact that the elements comprising an effective immune response directed against HIV are poorly understood. Simian immunodeficiency virus (SIV) infection of Mauritian cynomolgus macaques (MCM) provides the best model for unraveling the correlates of protection against SIV. With SIV infection of MCM, we can select the timing, route, dose, and sequence of the infecting virus. Additionally, the limited genetic diversity of MCM facilitates selection of genetically matched individuals that can be monitored throughout the acute phase of infection and enables more frequent and invasive sampling, especially of mucosal sites.Macaques infected with live-attenuated SIV, like SIVmac239Δnef, exhibit robust protection from pathogenic SIV infection (8, 10, 20, 25, 29, 35, 40, 44, 49, 53). While previous studies have included considerable heterogeneity in the strain of attenuated SIV, challenge strain, and macaque species used, they demonstrate collectively the broad spectrum of attenuated SIVs that effectively protect macaques against pathogenic challenge. Several studies have also shown that attenuated SIV effectively protects cynomolgus macaques against pathogenic challenge using an SIVmac239C8 virus (1-3). Like SIVmac239Δnef, this virus has a deletion in the nef gene, but this deletion is a considerably smaller 12-bp deletion than the 182-bp deletion in SIVmac239Δnef (24). Importantly, there are no studies that establish the protective efficacy of SIVmac239Δnef in MCM. Nevertheless, peak SIVmac239Δnef viral loads in MCM parallel the range established in rhesus macaques, between 3.2 × 10³ and 9.4 × 10⁵ (35), but fall below peak loads established in a separate study (8). The differences observed between the rhesus macaque study and our own could be due to differences in challenge dose. Long-term control of SIVmac239Δnef in MCM is also similar to that in rhesus macaques (35). It is critical to understand why live-attenuated SIV vaccines are so effective, with the ultimate goal of using these principals to develop a vaccine that is safe for use in humans.There are several plausible explanations for why live-attenuated SIV vaccines provide effective protection against challenge with pathogenic SIV. These explanations range from viral interference to a robust vaccine-elicited immune response (19, 43, 45, 47). We currently understand several aspects of live-attenuated vaccination with SIVmac239Δnef. First, there is an inverse relationship between the degree of attenuation and the level of protection (21). This relationship suggests that vigorous viral replication is important for the generation of an effective anti-SIV immune response. Second, the greater the sequence diversity between the vaccine strain and the challenge strain, the weaker is the protection provided by the vaccine (53). This demonstrates that an adaptive immune response that recognizes similar epitopes or virus features between the vaccine and challenge strains is necessary for protection in this model. Finally, vaccination with live-attenuated SIV requires a 15- to 20-week induction phase to achieve protection in the majority of animals (8). Thus, there is a direct relationship between the time postvaccination and the degree of protection between 0 and 20 weeks postvaccination. This temporal relationship suggests that a fully developed memory response is required to protect against pathogenic SIV challenge. Together, these observations argue that SIV-specific CD8⁺ T-lymphocyte responses might be important in protection as these responses would be less useful in the setting of heterologous virus challenge and require both robust viral replication and time to develop. Such responses, however, can be weak to nonexistent in the blood of vaccinated animals and frequently do not correlate with disease progression, leading some investigators to question their importance in protective immunity (25, 29, 45, 46). We hypothesize that continued replication of live-attenuated SIV in the mucosal tissues may lead to effective, compartmentalized memory T-cell responses that are important in controlling pathogenic SIV challenge.It is possible that prophylactic mucosal immunity is required to prevent viral replication soon after SIV infection and to minimize the destruction of mucosal immune cells that occurs within the first 3 weeks of SIV infection (9, 22, 26, 30). Initial depletion of effector memory CD4⁺ T cells in the gut-associated lymphoid tissue (GALT) combined with continuous viral replication leads to prolonged immune activation, eventual depletion of central memory CD4⁺ T cells, and the development of AIDS. An effective mucosal immune response elicited by live-attenuated SIV vaccination may prevent the initial CD4⁺ T-cell depletion from the gut. Several recent studies confirm a critical protective role for CD8⁺ T cells in the genital tract after vaccination with SHIV89.6, demonstrating that a mucosal immune response is capable of protecting against or ameliorating SIV infection (14-16, 48). Another study has also demonstrated the presence of high-frequency, polyfunctional T-cell responses in the mucosal tissues of elite controllers, i.e., individuals who maintain plasma viral loads below 75 copies/ml, compared to blood from the same individual, tissues of noncontrollers, and antiretroviral drug-treated patients. This study also provides a correlation between mucosal CD8⁺ T-cell responses and HIV control (12).While studying gut mucosal tissues is clearly an important part of understanding HIV/SIV pathology, there are several challenges to this undertaking. First, accessing gut tissues requires invasive sampling procedures, which are primarily limited to biopsy or time-of-death studies. Biopsies are often limited in number throughout the life span of an animal, while routine necropsy is cost-prohibitive for macaque studies. Second, these tissues are nonsterile. The digestive tract is teeming with floras that contaminate experiments requiring long-term cell culture. Finally, biopsies of mucosal tissues yield very few cells. These low cell numbers make ex vivo experiments very difficult or impossible to perform. Investigators have developed techniques to assess gut mucosal lymphocyte function by expanding these cells in vitro under sterile conditions with antibiotics and then using them in enzyme-linked immunospot assays (ELISPOT) or intracellular cytokine secretion (ICS) assays (18, 42). However, these experiments still suffer from two problems: (i) cells are altered in vitro, which may change their functional capacity; and, (ii) these experiments still rely on indirect measures of CD8⁺ T-cell function. These difficulties have limited research on mucosal CD8⁺ T-cell immunity during SIV infection.In light of these challenges, we decided to focus on the lung mucosal tissue, using CD8⁺ T cells isolated from bronchoalveolar lavage fluid (BAL). BAL samples lung mucosa where there are a large number of resident lymphocytes that encounter respiratory pathogens. Furthermore, BAL provides a minimally invasive sampling of a mucosal tissue that can be performed frequently, and BAL harbors effector T cells similar to GALT (32). These factors make the lung an ideal site for sampling mucosal CD8⁺ T cells.We modified an ex vivo viral suppression assay that tests the ability of CD8⁺ T cells to prevent viral replication in MCM (7, 27, 28, 36, 51, 54, 55). Using this approach, we compared the suppressive capacity of CD8⁺ T cells isolated from lung and blood, and we found that CD8⁺ T cells from the lung are more effective at suppressing viral replication than CD8⁺ T cells from the blood. This assay does not manipulate lung lymphocytes in vitro and provides a direct measure of CD8⁺ T-cell function. Furthermore, our data support the idea that CD8⁺ T cells in blood and mucosal tissue are not functionally equivalent, that blood lymphocytes are not a perfect surrogate for mucosal lymphocytes, and that mucosal T cells attenuate SIV replication to a greater extent than blood T cells.     

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.     

Dominant CD8+ T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8⁺ T-lymphocyte responses in Mamu-A*01⁺ rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01⁺ rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8⁺ T-lymphocyte response in the absence of secondary Gag p11C-specific CD8⁺ T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8⁺ T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8⁺ T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.CD8⁺ T lymphocyte responses play a central role in controlling human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) infections in nonhuman primates (18, 20, 29, 41). Naturally occurring virus-specific CD8⁺ T-lymphocyte responses typically focus on a limited number of dominant epitopes (52). However, accumulating data indicate that a broad cellular immune response, in which multiple viral epitopes are recognized by CD8⁺ T lymphocytes, confers better protection against viral replication than a restricted cellular immune response (26, 33). Therefore, it has been suggested that increasing the magnitude of subdominant epitope-specific responses may increase the breadth of a cellular immune response and provide enhanced protection against HIV/SIV replication.An understanding of the factors that influence the immunodominance hierarchy of viral epitopes will be needed to develop vaccination strategies that can generate the greatest breadth of virus-specific CD8⁺ T-lymphocyte responses. Differences in antigen processing, competition between epitope peptides for major histocompatibility complex (MHC) class I molecules, T-cell receptor (TCR) repertoire, TCR affinity for peptide class I complexes, and immunodomination have been shown to contribute to the dominance of an epitope-specific response (6, 10, 24, 32, 45, 52). In addition, studies have shown that immunodominance patterns for T-lymphocyte epitopes may differ following a primary and secondary exposure to the same viral antigen (4, 5, 43).In the present study, we observed that Mamu-A*01⁺ rhesus monkeys primed with plasmid DNA and boosted with recombinant adenovirus (rAd) vaccines encoding SIVmac239 Gag-Pol-Nef and HIV-1 Env proteins generated Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses of comparable magnitude. However, while there was a significant expansion of Gag p11C-specific CD8⁺ T-lymphocyte populations following challenge with pathogenic simian-human immunodeficiency virus 89.6P (SHIV-89.6P), there was no significant expansion of the Env p41A-specific CD8⁺ T-lymphocyte populations. We hypothesized that factors influencing the relative immunodominance of the primed Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses after viral challenge may have contributed to the observed differences in their secondary expansion. In the present study, we sought to identify the potential factors contributing to this immunodominance.     

Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV_mac239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4⁺ T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV_mac239. However, significantly lower viral loads and higher memory CD4⁺ T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV_mac239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.     

FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeficiency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load

The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25⁺ FoxP3⁺ regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4⁺ regulatory T cells have been studied in the most detail, but CD8⁺ CD25⁺ FoxP3⁺ T cells also have regulatory properties. We monitored the dynamics of CD25⁺ FoxP3⁺ T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4⁺ CD25⁺ FoxP3⁺ T cells paralleled that of memory CD4⁺ T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25⁺ FoxP3⁺ T cells was observed in peripheral lymph nodes. A small number of CD4⁺ CD25⁺ FoxP3⁺ T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8⁺ CD25⁺ FoxP3⁺ T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4⁺ T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8⁺ CD25⁺ FoxP3⁺ T cells being indicative of a poor prognosis.     

Characterization of SIV-specific CD4+ T-helper proliferative responses in macaques immunized with live-attenuated SIV

Analysis of immune responses generated by live-attenuated simian immunodeficiency virus (SIV) strains may provide clues to the mechanisms of protective immunity induced by this approach. We examined SIV-specific T-helper responses in macaques immunized with the live-attenuated SIV strains SIVmac239deltanef and SIVmac239delta3. Optimization of the concentration and duration of antigenic stimulation resulted in the detection of relatively strong SIV-specific proliferative responses, with peak stimulation indices of up to 84. SIV-specific proliferative responses were mediated by CD4+ T cells and were major histocompatibility (MHC) class II restricted. Limiting dilution analysis revealed SIV-specific T-helper precursor frequencies of up to 96 per 10(6) peripheral blood mononuclear cells (PBMC). Intracellular flow-cytometric analysis demonstrated the production of interleukin (IL)-2, interferon (IFN)-gamma, RANTES and macrophage inhibitory protein-1alpha (MIP-1alpha) by T lymphocytes from SIVmac239deltanef-vaccinated animals following SIV p55 stimulation. Induction of strong SIV-specific T-helper responses by live-attenuated SIV vaccines may play a role in their ability to induce protective immunity.     

Dynamics of T- and B-lymphocyte turnover in a natural host of simian immunodeficiency virus

Increased lymphocyte turnover is a hallmark of pathogenic lentiviral infection. To investigate perturbations in lymphocyte dynamics in natural hosts with nonpathogenic simian immunodeficiency virus (SIV) infection, the nucleoside analog bromodeoxyuridine (BrdU) was administered to six naturally SIV-infected and five SIV-negative sooty mangabeys. As a measure of lymphocyte turnover, we estimated the mean death rate by fitting a mathematical model to the fraction of BrdU-labeled cells during a 2-week labeling and a median 10-week delabeling period. Despite significantly lower total T- and B-lymphocyte counts in SIV-infected sooty mangabeys than in SIV-negative mangabeys, the turnover rate of B lymphocytes and CD4⁺ and CD8⁺ T lymphocytes was not increased in the SIV-infected animals. A small, rapidly proliferating CD45RA⁺ memory subset and a large, slower-proliferating CD45RA⁻ central memory subset of CD4⁺ T lymphocytes identified in the peripheral blood of sooty mangabeys also did not show evidence of increased turnover in the context of SIV infection. Independently of SIV infection, the turnover of CD4⁺ T lymphocytes in sooty mangabeys was significantly higher (P < 0.01) than that of CD8⁺ T lymphocytes, a finding hitherto not reported in rhesus macaques or humans. The absence of aberrant T-lymphocyte turnover along with an inherently high rate of CD4⁺ T-lymphocyte turnover may help to preserve the pool of central memory CD4⁺ T lymphocytes in viremic SIV-infected sooty mangabeys and protect against progression to AIDS.     

Reduction of peak viremia by an integration-defective SIV proviral DNA vaccine in rhesus macaques

An integrase-defective SIV (idSIV) vaccine delivered by a DNA prime and viral particle boost approach can suppress viral loads (VLs) during the acute infection stage after intravenous SIVmac239 challenge. This study investigated how idSIV DNA and viral particle immunization alone contributed to the suppression of VLs in Chinese rhesus macaques after SIV challenge. Two macaques were immunized with idSIV DNA five times and two macaques were immunized with idSIV viral particles three times. Cellular and humoral immune responses were measured in the vaccinated macaques after immunization. The VLs and CD4⁺ T cell counts were monitored for 28 weeks after the intravenous SIVmac239 challenge. The SIV-specific T cell responses were only detected in the DNA-vaccinated macaques. However, binding and neutralizing antibodies against autologous and heterologous viruses were moderately better in macaques immunized with viral particles than in macaques immunized with DNA. After the challenge, the mean peak viremia in the DNA group was 2.3 logs lower than that in the control group, while they were similar between the viral particle immunization and control groups. Similar CD4⁺ T cell counts were observed among all groups. These results suggest that idSIV DNA immunization alone reduces VLs during acute infection after SIV challenge in macaques and may serve as a key component in combination with other immunogens as prophylactic vaccines.     

Live-Attenuated Lentivirus Immunization Modulates Innate Immunity and Inflammation while Protecting Rhesus Macaques from Vaginal Simian Immunodeficiency Virus Challenge

Immunization with attenuated lentiviruses is the only reliable method of protecting rhesus macaques (RM) from vaginal challenge with pathogenic simian immunodeficiency virus (SIV). CD8(+) lymphocyte depletion prior to SIVmac239 vaginal challenge demonstrated that a modest, Gag-specific CD8(+) T cell response induced by immunization with simian-human immunodeficiency virus 89.6 (SHIV89.6) protects RM. Although CD8(+) T cells are required for protection, there is no anamnestic expansion of SIV-specific CD8(+) T cells in any tissues except the vagina after challenge. Further, SHIV immunization increased the number of viral target cells in the vagina and cervix, suggesting that the ratio of target cells to antiviral CD8(+) T cells was not a determinant of protection. We hypothesized that persistent replication of the attenuated vaccine virus modulates inflammatory responses and limits T cell activation and expansion by inducing immunoregulatory T cell populations. We found that attenuated SHIV infection decreased the number of circulating plasmacytoid dendritic cells, suppressed T cell activation, decreased mRNA levels of proinflammatory mediators, and increased mRNA levels of immunoregulatory molecules. Three days after SIV vaginal challenge, SHIV-immunized RM had significantly more T regulatory cells in the vagina than the unimmunized RM. By day 14 postchallenge, immune activation and inflammation were characteristic of unimmunized RM but were minimal in SHIV-immunized RM. Thus, a modest vaccine-induced CD8(+) T cell response in the context of immunoregulatory suppression of T cell activation may protect against vaginal HIV transmission.     

Simian immunodeficiency virus (SIV) infection influences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIV-infected sooty mangabeys

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM, but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition, immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides, there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM, their role in disease resistance in SM remains unclear.     

Early divergence in lymphoid tissue apoptosis between pathogenic and nonpathogenic simian immunodeficiency virus infections of nonhuman primates

The events that contribute to the progression to AIDS during the acute phase of a primate lentiviral infection are still poorly understood. In this study, we used pathogenic and nonpathogenic simian models of simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) and African green monkeys (AGMs), respectively, to investigate the relationship between apoptosis in lymph nodes and the extent of viral replication, immune activation, and disease outcome. Here, we show that, in SIVmac251-infected RMs, a marked increased in lymphocyte apoptosis is evident during primary infection at the level of lymph nodes. Interestingly, the levels of apoptosis correlated with the extent of viral replication and the rate of disease progression to AIDS, with higher apoptosis in RMs of Indian genetic background than in those of Chinese origin. In stark contrast, no changes in the levels of lymphocyte apoptosis were observed during primary infection in the nonpathogenic model of SIVagm-sab infection of AGMs, despite similarly high rates of viral replication. A further and early divergence between SIV-infected RMs and AGMs was observed in terms of the dynamics of T- and B-cell proliferation in lymph nodes, with RMs showing significantly higher levels of cycling cells (Ki67⁺) in the T-cell zones in association with relatively low levels of Ki67⁺ in the B-cell zones, whereas AGMs displayed a low frequency of Ki67⁺ in the T-cell area but a high proportion of Ki67⁺ cells in the B-cell area. As such, this study suggests that species-specific host factors determine an early immune response to SIV that predominantly involves either cellular or humoral immunity in RMs and AGMs, respectively. Taken together, these data are consistent with the hypotheses that (i) high levels of T-cell activation and lymphocyte apoptosis are key pathogenic factors during pathogenic SIV infection of RMs and (ii) low T-cell activation and apoptosis are determinants of the AIDS resistance of SIVagm-infected AGMs, despite high levels of SIVagm replication.