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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., Author Summary Annually, more than two million people worldwide are infected with HIV, the virus that causes AIDS. Rhesus macaques can be infected with SIV, a close relative and ancestor of HIV, resulting in simian AIDS, recapitulating key aspects of human HIV infection. SIVΔnef, a live attenuated form of SIV, protects rhesus macaques from subsequent challenge with pathogenic SIV and is widely viewed as the most effective SIV vaccine. Here we demonstrate that SIVΔnef persistence during the vaccination period drives both cell-mediated and humoral immune response maturation. During the vaccination period, cell-mediated immune responses elicited by SIVΔnef target more conserved regions of the virus rendering immune escape more difficult. Furthermore, the localization of the cell-mediated immune responses is shifted over time from peripheral blood to sites of viral production that are rich in uninfected SIV target cells, thereby positioning cell-mediated immune responses where they are most needed after wild-type SIV challenge. Similarly, SIVΔnef persistence during the vaccination period also leads to the accumulation and maturation of the humoral immune response. Our findings highlight the unique capacity of persistent vaccines to elicit durable and effective immune responses against wild-type SIV challenge.

Natural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A+NFIL3+RORC– and NKp44+NFIL3+RORC+. NKG2A+ NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44+ NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44+ NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44+ NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44+ NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

Natural killer (NK) cells contribute to control of HIV/SIV infection. We defined macaque NK-cell subsets based on expression of CD56 and CD16 and found their distribution to be highly disparate. CD16+ NK cells predominated in peripheral blood, whereas most mucosal NK cells were CD56+, and lymph nodes contained both CD56+ and CD16−CD56− (double-negative [DN]) subsets. Functional profiles were also distinct among subsets—CD16+ NK cells expressed high levels of cytolytic molecules, and CD56+ NK cells were predominantly cytokine-secreting cells, whereas DN NK possessed both functions. In macaques chronically infected with SIV, circulating CD16+ and DN NK cells were expanded in number and, although markers of cytoxicity increased, cytokine secretion decreased. Notably, CD56+ NK cells in SIV-infected animals up-regulated perforin, granzyme B, and CD107a. In contrast, the lymph node–homing molecules CD62 ligand (CD62L) and C-C chemokine receptor type 7 (CCR7), which are expressed primarily on CD56+ and DN NK cells, were significantly down-regulated on NK cells from infected animals. These data demonstrate that SIV infection drives a shift in NK-cell function characterized by decreased cytokine production, expanded cytotoxicity, and trafficking away from secondary lymphoid organs, suggesting that the NK-cell repertoire is not only heterogeneous but also plastic.

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses., Author Summary Natural killer (NK) cells recognize and kill infected cells without prior antigenic stimulation, and thus provide an important early defense against virus infection. NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. Inhibitory KIRs normally suppress NK cell responses through interactions with their MHC class I ligands on the surface of healthy cells. However, when these interactions are perturbed, this inhibition is lost resulting in NK cell activation and killing of the target cell. We investigated the functional implications of simian immunodeficiency virus (SIV) peptides bound by a common MHC class I molecule in the rhesus macaque that stabilize or disrupt binding to an inhibitory KIR. Whereas SIV peptides that stabilized KIR-MHC class I binding suppressed NK cell activation, peptides that disrupted this interaction did not and resulted in NK cell lysis. These findings demonstrate that viral peptides can modulate NK cell responses through KIR-MHC class I interactions, and are consistent with the possibility that human and simian immunodeficiency viruses may acquire changes in epitopes that increase the binding of MHC class I ligands to inhibitory KIRs as a mechanism to suppress NK cell responses.

We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.

Plasmacytoid dendritic cells (pDCs), a primary source of interferon α (IFN-α), provide a first line of innate immune defense against human immunodeficiency virus infection. However, their kinetics and functions during acute infection are poorly understood. In mucosal tissues of normal rhesus macaques, we found CD4(+) pDCs to be the subset responsible for most IFN-α and tumor necrosis factor α (TNF-α) production in response to Toll-like receptor (TLR) 7/8 stimulation, compared with relatively anergic CD4(-) pDCs. During acute simian immunodeficiency virus (SIV) infection, gut homing was imprinted on pDCs in the bone marrow, resulting in a decline in pDCs from circulation and secondary lymphoid tissues. Although the accumulated pDCs in the gut mucosae had robust cytokine responses to TLR7/8 stimulation in vitro, pDC gut migration occurred after infection and detection of SIV in plasma. Our data suggest that innate pDC responses do not control initial SIV seeding and dissemination but instead may contribute to ongoing immune activation in the gut.

Cytomegalovirus (CMV) is a herpesvirus establishing life-long persistence after primary infection. CMV infection can cause severe manifestations in immunocompromised subjects and fetuses but is usually asymptomatic in immunocompetent hosts. Data from several studies support an important role for CD4+ T lymphocytes in the control of human CMV infection. Early appearance of functional CMV-specific CD4+ T cells is associated with asymptomatic infections after kidney transplantation [1] and with a lower risk of viremia after hematopoietic stem cell transplantation [2]. In chronic infection, CMV-specific CD4+ T cells acquire direct antiviral functions and are pivotal for optimal B and CD8+ T-lymphocyte responses [3]. CMV-specific CD4+ T cells produce multiple cytokines, including interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and interleukin 2 (IL-2), have potent proliferative capacity and can display major histocompatibility complex class II–restricted cytotoxicity [4, 5]. Asymptomatic primary CMV infection in immunocompetent hosts, particularly children, often leads to virus shedding in bodily secretions for a prolonged duration, suggesting incomplete immune control [6, 7]. Following primary infection, CMV-specific CD4+ T cells emerge rapidly but acquire the capacity to proliferate in vitro in response to viral antigens only several months after seroconversion [1, 7, 8]. While the delayed acquisition of functional CD4+ T-lymphocyte responses in primary infection is likely to contribute to the incomplete control of CMV replication, the etiology of functional CD4+ T-lymphocyte exhaustion remains uncertain. We recently observed that the slow acquisition of proliferative responses in primary CMV infection is associated with a reduced capacity to produce cytokines, including IL-2, IFN-γ, and TNF-α [9]. Clinical studies suggest that this functional impairment is a risk factor for mother-to-fetus transmission of CMV and for CMV retinitis in human immunodeficiency virus (HIV)–infected patients [10–13]. Thus, development of therapeutic strategies to prevent the clinical complications associated with primary CMV infection will require a better understanding of the mechanisms compromising its immune control. Rhesus macaques are currently the best available animal model of human CMV infection [14]. CMV infection is widely prevalent among captive group-housed rhesus macaques, and rhesus CMV shares close genetic similarity to human CMV [15–17]. Similar to humans, rhesus CMV infection is asymptomatic in immunocompetent macaques, and CMV reactivation-associated disease manifests only in the setting of immunosuppression [18–20]. In contrast to chronic infection, there are limited data on the virologic and immunologic events in naturally acquired primary rhesus CMV infection. We and others have shown that maternal antibodies to CMV wane around 1 year of age and that seroconversion following natural infection occurs soon thereafter [15, 18]. Furthermore, hand-reared newborn macaques separated at birth from their mothers remain CMV seronegative after loss of maternal CMV antibodies [18], suggesting that natural CMV acquisition occurs in the postnatal period. In this study, we investigated the relationship between viral load and CMV-specific CD4+ T-lymphocyte responses at different time points following natural CMV acquisition in rhesus macaques.

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34+ bone marrow cells transduced with a retroviral vector expressing eGFP. CD34+ cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow–derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8+lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34+ cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34+ hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol.

The identification of MHC class I ligands for rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to our basic understanding of KIR and MHC class I coevolution and to the study of NK cell responses in this nonhuman primate model for AIDS and other viral diseases. In this study, we show that Mamu-KIR3DL01, which is expressed by ∼90% of rhesus macaques, recognizes MHC class I molecules with a Bw4 motif. Primary NK cells expressing Mamu-KIR3DL01 were identified by staining with a mAb which, in this study, was shown to bind Mamu-KIR3DL01 allotypes with an aspartic acid at position 233. The cytolytic activity of Mamu-KIR3DL01+ NK cells was suppressed by cell lines expressing the Bw4 molecules Mamu-B*007:01, -B*041:01, -B*058:02, and -B*065:01. The Bw4 motif was necessary for Mamu-KIR3DL01 recognition because substitutions in this region abrogated Mamu-KIR3DL01+ NK cell inhibition. However, the presence of a Bw4 motif was not sufficient for recognition because another Bw4 molecule, Mamu-B*017:01, failed to suppress the cytolytic activity of these NK cells. Replacement of three residues in Mamu-B*017:01, predicted to be KIR contacts based on the three-dimensional structure of the human KIR3DL1-HLA-Bw4 complex, with the corresponding residues at these positions for the other Mamu-Bw4 ligands restored Mamu-KIR3DL01+ NK cell inhibition. These results define the ligand specificity of one of the most polymorphic and commonly expressed KIRs in the rhesus macaque and reveal similarities in Bw4 recognition by Mamu-KIR3DL01 and human KIR3DL1, despite the absence of an orthologous relationship between these two KIRs or conservation of surface residues predicted to interact with MHC class I ligands.

SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1–3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques.

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.

Despite considerable research, the mechanisms by which HIV disrupts thymic function remain controversial. We have described the phenotypic changes that occur in the thymus of SIV-infected macaques during acute SIV infection. In this study, we analyzed the effects of SIV infection on apoptotic pathways in thymic tissue from newborn macaques infected with SIV. Thymocyte apoptosis was accompanied by a modest increase in surface Fas expression, a profound decrease in the frequency of bcl-2-positive cells, as well as the amount of bcl-2 per cell. With control of viral replication, levels of bcl-2 and Fas returned to baseline together with a return to basal levels of apoptosis. In the thymus, SIV infection resulted in depletion of CD4+CD8+ thymocytes, an increase in apoptosis of thymocytes, and a down-regulation of MHC class I molecules. These changes peaked 14–21 days after infection at or just after peak viremia. This data further suggests disruption of the antiapoptotic pathway regulated by bcl-2 plays a critical role in SIV-induced apoptosis of thymocytes.