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The live attenuated vaccine (LAV) SIVmac239Δnef (SIVΔnef) confers the best protection among all the vaccine modalities tested in rhesus macaque model of HIV-1 infection. This vaccine has a unique feature of time-dependent protection: macaques are not protected at 3-5 weeks post vaccination (WPV), whereas immune protection emerges between 15 and 20 WPV. Although the exact mechanisms of the time-dependent protection remain incompletely understood, studies suggested that both cellular and humoral immunities contribute to this time-dependent protection. To further elucidate the mechanisms of protection induced by SIVΔnef, we longitudinally compared the global gene expression profiles of SIV Gag-CM9+ CD8+ (Gag-specific CD8+) T cells from peripheral blood of Mamu-A*01+ rhesus macaques at 3 and 20 WPV using rhesus microarray. We found that gene expression profiles of Gag-specific CD8+ T cells at 20 WPV are qualitatively different from those at 3 WPV. At 20 WPV, the most significant transcriptional changes of Gag-specific CD8+ T cells were genes involved in TCR signaling, differentiation and maturation toward central memory cells, with increased expression of CCR7, TCRα, TCRβ, CD28 and decreased expression of CTLA-4, IFN-γ, RANTES, granzyme A and B. Our study suggests that a higher quality of SIV-specific CD8+ T cells elicited by SIVΔnef over time contributes to the maturation of time-dependent protection.

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination., Competing Interests: The authors have declared that no competing interests exist.

Although immune pressure exerted by MHC class I-restricted cytotoxic T lymphocytes (CTL) are an important determinant of outcome in pathogenic HIV and SIV infection, lack of data on MHC class I genes has hampered study of its role in natural hosts with nonpathogenic SIV infection. In this study, we cloned and characterized full-length MHC class I genes derived from the cDNA library of two unrelated naturally infected sooty mangabeys (Cercocebus atys) in whom SIV-specific CTL epitopes were previously mapped. Twenty one full-length MHC class I alleles consisting of five MHC-A (Ceat-A), 13 MHC-B (Ceat-B), and three MHC-E (Ceat-E) alleles were identified. Sequence-specific primers (SSP) for high-throughput screening of genomic DNA by PCR were developed for 16 of the 18 Ceat-A and Ceat-B alleles. Screening of 62 SIV-negative and 123 SIV-infected sooty mangabeys at the Yerkes National Primate Research Center (YNPRC) revealed the presence of up to four MHC-A and eight MHC-B alleles in individual mangabeys, indicating that similar to macaque species, mangabeys have at least two duplications of the MHC-A locus and four duplications of the MHC-B locus in the absence of an MHC-C locus. Using stable transfectants of Ceat MHC Class I alleles in the MHC-null 721.221 cell line, we identified Ceat-B*12:01 as the restricting allele of a previously reported Nef20-28 CTL epitope. Ceat-B*1201/Nef20-28 tetramers identified tetramer-positive CD8+ T lymphocytes in Ceat-B*1201-positive SIV-infected mangabeys. This study has laid the groundwork for comprehensive analysis of CTL and SIV evolution in a natural host of SIV infection.

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states.

The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.

Problem: Mucosal T lymphocyte responses in the female reproductive tract, the primary site of HIV transmission in women, may be critical for initial control of virus infection. In addition, characterization of genital immune responses to HIV will be important for the development of a vaccine capable of preventing infection by this route., Method of Study: We analyzed lymphocytes isolated from vagina and cervix of chronically SIV-infected macaques for the frequency of SIV Gag tetramer-binding cells and expression of chemokine receptors., Results: We found that the frequency of SIV-specific CD8+ T cell responses was 3- to 30-fold higher in genital tissues than in peripheral blood. SIV-specific CD8+ T cells in genital tissues expressed high levels of CXCR3 and CCR5, chemokine receptors normally expressed on memory T cells that home to inflamed tissues. Cells expressing CXCR3 colocalized with its chemokine ligand CXCL9 [monokine induced by interferon gamma, MIG] in the vaginal lamina propria., Conclusion: These results indicate that the frequency of SIV-specific CD8+ T cells in the female genital mucosa is enriched compared with peripheral blood and provide initial information regarding the signals that direct recruitment of T cells to the female reproductive tract., (© 2011 John Wiley & Sons A/S.)

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8(+) T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8(+) T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8(+) T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8(+) T cells to suppress viral replication from SIVmac239-infected CD4(+) T cells. Using this assay, we established an antiviral hierarchy when we compared CD8(+) T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8(+) T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8(+) T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8(+) T-cell response may be comprised of several factors.

Vaccinia virus (VACV) induces a vigorous virus-specific CD8+ T cell response that plays an important role in control of poxvirus infection. To identify immunodominant poxvirus proteins and to facilitate future testing of smallpox vaccines in non-human primates, we used an algorithm for the prediction of VACV peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 VACV ORFs; 100 of these peptides did not contain the canonical proline at position three of the Mamu-A*01 binding motif. Cellular immune responses in PBMC from two vaccinia-vaccinated Mamu-A*01+ macaques were assessed by IFNgamma ELISPOT assays. Vaccinated macaques recognized 17 peptides from 16 different ORFs with 6 peptides recognized by both macaques. Comparison with other orthopoxvirus sequences revealed that 12 of these epitopes are strictly conserved between VACV, variola, and monkeypoxvirus. ELISPOT responses were also observed to eight epitopes that did not contain the canonical P3 proline. These results suggest that the virus-specific CD8+ T cell response is broadly directed against multiple VACV proteins and that a subset of these T cell epitopes is highly conserved among orthopoxviruses.

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.

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.

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-gamma) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-gamma responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.

The ability of memory T cells to mount a recall response plays a key role in the ability of vaccinated animals to contain viral challenge. In this study, we intensively monitored the expansion of SIV Gag-specific CD8+ T cells in peripheral blood and tissues of rhesus macaques vaccinated with the attenuated strain SIVmac239Delta3 and challenged with the pathogenic viruses SIVmac239 or SIVsmE660. Although all vaccinated animals were infected with challenge virus, peak levels of plasma viremia in vaccinees were decreased by 1.5 to 2 logs as compared with naive controls. Decreased levels of plasma viremia in vaccinated animals were evident as early as 7 days post-challenge, well before the expansion of SIV-specific CD8+ T cells. Expansion of SIV-specific CD8+ T cells was not observed in peripheral blood or tissues until at least 14 days after infection and did not occur in most animals until after the initial peak of viral replication. The observation that expansion of SIV-specific CD8+ T cells is delayed until 7 days or more after initial detection of viremia highlights fundamental limitations in the ability of lentivirus-specific CD8+ T cells to mediate protection against challenge.

Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4(+) and CD8(+) T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8(+) T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4(+) T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8(+) T lymphocytes associated with SIV-induced impairment of CMV-specific CD4(+) T-cell help.

Immunization of macaques with attenuated simian immunodeficiency virus (SIV) with deletions in nef (SIVΔnef) is shown to elicit protective immunity to infection by pathogenic SIV, yet the mechanisms that orchestrate protection and prevent pathogenesis remains unknown. We utilized whole-genome transcriptional profiling to reveal molecular signatures of protective immunity in circulating CD8+ T cells of rhesus macaques vaccinated with SIVmac239Δnef and challenged with pathogenic SIVmac251. Our findings suggest that protective immunity to pathogenic SIV infection induced by SIVmac239∆nef is associated with balanced induction of T cell activation and immunoregulatory mechanisms and dampened activation of interferon-induced signaling pathways and cytolytic enzyme production as compared with pathogenic SIVmac251 infection of unvaccinated controls. We provide evidence that protective immunity to SIVmac251 correlates with induction of biomarkers of T cell activation, differentiation, signaling, and adhesion that were down regulated in unvaccinated controls. The study highlights potential immunomodulatory networks associated with protective immunity against the virus.