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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., Competing Interests: The authors have declared that no competing interests exist.

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.

Multiple studies suggest that plasmacytoid dendritic cells (pDCs) are depleted and dysfunctional during human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but little is known about pDCs in the gut-the primary site of virus replication. Here, we show that during SIV infection, pDCs were reduced 3--fold in the circulation and significantly upregulated the gut-homing marker α4β7, but were increased 4-fold in rectal biopsies of infected compared to naive macaques. These data revise the understanding of pDC immunobiology during SIV infection, indicating that pDCs are not necessarily depleted, but instead may traffic to and accumulate in the gut mucosa.

We evaluated the potential of an anti-human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4(+) cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4(+) T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4(+) cell. Upon reinfusion of engineered autologous CD4(+) cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4(+) T cells progressively decreased, concurrent with loss of CD4(+) cells and elevated viral loads in both animals. However, CD4(+) T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4(+) cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo.

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, P<0.0001). 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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

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.

Background: Pathogenic HIV and SIV infections characteristically deplete central memory CD4(+) T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4(+) T-cell play roles in protection against wild-type virus challenge., Methods: Rhesus macaques were vaccinated with SIVmac239Deltanef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies., Results: Animals vaccinated with SIVmac239Deltanef demonstrated no loss of CD4(+) T cells in any tissue, and in fact CCR5(+) and CD28(+)CD95(+) central memory CD4(+) T cells were significantly increased. In contrast, CD4(+) T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Deltanef vaccination then declined following primary infection., Conclusion: We demonstrated in this study that SIVmac239Deltanef vaccination did not deplete CD4(+) T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4(+) T cells did not appear to influence protective immunity.

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated., Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences., Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

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.