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The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to an HIV/SIV cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCFs) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of NK cells in macaque lymph nodes that expressed BCF-homing receptor CXCR5 and accumulated within BCFs during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g., perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular Th cells that comprise a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared with the CXCR5- subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, and this may have important implications for HIV cure strategies.

Dysfunction of virus-specific CD4 + T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4 + T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (T FH cell)-like profile characterized HIV-specific CD4 + T cells in viremic infection. HIV-specific CD4 + T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the T H 1, T H 17 and T H 22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with T FH cells, but persistently low expression of genes associated with T H 1, T H 17 and T H 22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4 + T cells and involves both gain of function and loss of function.

Live attenuated vaccines such as SIV with a deleted nef gene have provided the most robust protection against subsequent vaginal challenge with wild-type (WT) SIV in the SIV-rhesus macaque model of HIV-1 transmission to women. Hence, identifying correlates of this protection could enable design of an effective HIV-1 vaccine. One such prechallenge correlate of protection from vaginal challenge has recently been identified as a system with three components: 1) IgG Abs reacting with the viral envelope glycoprotein trimeric gp41; 2) produced by plasma cells in the submucosa and ectopic tertiary lymphoid follicles in the ectocervix and vagina; and 3) concentrated on the path of virus entry by the neonatal FcR in the overlying epithelium. We now examine the mucosal production of the Ab component of this system after vaginal challenge. We show that vaginal challenge immediately elicits striking increases in plasma cells not only in the female reproductive tract but also at other mucosal sites, and that these increases correlate with low but persistent replication at mucosal sites. We describe vaginal ectopic follicles that are structurally and functionally organized similar to follicles in secondary lymphoid organs, and we provide inferential evidence for a key role of the female reproductive tract epithelium in facilitating Ab production, affinity maturation, and class switch recombination. Vaccination thus accesses an epithelial-immune system axis in the female reproductive tract to respond to exposure to mucosal pathogens. Designing strategies to mimic this system could advance development of an effective HIV-1 vaccine., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Engagement of early stage investigators (ESIs) in the search for a safe and effective vaccine is critical to the success of this highly challenging endeavor. In the wake of disappointing results from a large-scale efficacy trial, the HIV Vaccine Trials Network (HVTN) and Center for HIV/AIDS Vaccine Immunology (CHAVI) developed a novel mentored research program focused on the translation of findings from nonhuman primate studies to human trials of experimental vaccines. From 2008 to 2011, 14 ESI Scholars were selected from 42 complete applications. Post program surveys and tracked outcomes suggest that the combination of flexible funding, transdisciplinary mentorship, and structured training and networking promoted the scientific contributions and career development of promising ESIs. Embedding a multicomponent research program within collaborative clinical trial networks and research consortia is a promising strategy to attract and retain early career investigators and catalyze important translational science., (© 2014 Wiley Periodicals, Inc.)

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker alpha4beta7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, alpha4beta7 expression was associated with increased NK cell activation and, on CD16(+) NK cells, delineated a unique dual-function cytotoxic-CD107a(+)/gamma interferon (IFN-gamma)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56(+) NK cells, alpha4beta7(+) and alpha4beta7(-) NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.

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.

Ongoing antigenic stimulation appears to be an important prerequisite for the persistent expression of programmed death 1 (PD-1), an inhibitory TCR coreceptor of the CD28 family. Although recent publications have emphasized the utility of PD-1 as a marker for dysfunctional T cells in chronic viral infections, its dependence on antigenic stimulation potentially renders it a sensitive indicator of low-level viral replication. To explore the antigenic threshold for the maintenance of PD-1 expression on virus-specific T cells, we compared PD-1 expression on virus-specific and memory T cell populations in controlled and uncontrolled SIV and HIV-1 infection. In both controlled live attenuated SIV infection in rhesus macaques and HIV-1 infection in elite controllers, elevated levels of PD-1 expression were observed on SIV- and HIV-1-specific CD8(+) T cells. However, in contrast to chronic wild-type SIV infection and uncontrolled HIV-1 infection, controlled SIV/HIV-1 infection did not result in increased expression of PD-1 on total memory T cells. PD-1 expression on SIV-specific CD8(+) T cells rapidly decreased after the emergence of CTL escape in cognate epitopes, but was maintained in the setting of low or undetectable levels of plasma viremia in live attenuated SIV-infected macaques. After inoculation of naive macaques with a single-cycle SIV, PD-1 expression on SIV-specific CD8(+) T cells initially increased, but was rapidly downregulated. These results demonstrate that PD-1 can serve as a sensitive indicator of persistent, low-level virus replication and that generalized PD-1 expression on T lymphocytes is a distinguishing characteristic of uncontrolled lentiviral infections.

The rhesus macaque model is a useful experimental system to evaluate effects of T-cell autotransfusion and gene therapies for HIV-1 infection and AIDS prior to a clinical trial. To obtain sufficient numbers of primary macaque CD4 T lymphocytes for this purpose, we examined the culture conditions that were needed to optimize ex vivo activation and expansion of macaque primary CD4-enriched peripheral blood mononuclear cells (PBMCs). In this report, we compared the effects of various stimulants on cell expansion, surface expression of CCR5 and CXCR4, and levels of transduction with a Moloney leukemia virus (MoLV) vector encoding the phenotypic selection marker truncated human nerve growth factor receptor (deltaNGFR) alone or with the human anti-HIV-1 tat intrabody sFvhutat2. The use of feeder cells strikingly increased the proliferation rate of macaque CD4-enriched PBMCs in vitro. In the presence of an irradiated rhesus macaque B-lymphoblastoid cell line (BLCL), the highest cell expansion over 21 days was achieved with cells activated by Con A (9648-fold), in turn, from high to low, phytohemagglutinin (PHA) (4855-fold), and anti-CD3/CD28-coated beads (2367-fold). Further studies showed that BLCL feeder cells were more effective than human PBMCs (hPBMCs) in promoting proliferation of macaque CD4-enriched PBMCs activated with Con A and anti-CD3/CD28, respectively. The combined use of both BLCL and hPBMC feeder cells did not further increase cell expansion when compared with the use of BLCL cells alone. In addition, the addition of BLCL-conditioned medium (CM) and hPBMC-CM induced cell growth at a rate higher than did the culture medium alone but not as high as with feeder cells. Con A-activated macaque CD4-enriched PBMCs retained 88% of CXCR4 and 39% of CCR5 expression over 17 days compared with PHA-activated cells (50% for CXCR4, 16% for CCR5) and anti-CD3/CD28-activated cells (34% for CXCR4, 37% for CCR5). Finally, PHA, Con A, and CD3/CD28-coated beads supported comparable levels of MoLV transduction. The results should improve the utility of the rhesus macaque model for the testing of T-cell autotransfusion and gene therapies for HIV-1 infection/AIDS.

Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression.ImportanceNHP models of HIV infection constitute a powerful tool with which to study viral pathogenesis in order to gain critical information for a better understanding of HIV infection in humans. Here we studied progressive and nonprogressive simian immunodeficiency virus (SIV) infection models in both natural and nonnatural host NHP species. Regardless of the pathogenicity of the virus infection and regardless of the NHP species studied, the magnitude of viremia, as measured by area under the curve, during the first 4 weeks of infection correlated positively with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between nonprogressive and progressive SIV infection models. The qualitative differences in the early immune response in pathogenic and nonpathogenic infections identified here may be important determinants of the subsequent disease course.

The article reports that HIV and the related simian immunodeficiency virus cause AIDS by infecting the master regulatory cells of the immune system--T helper cells, better known as CD4+ T cells. It is generally years before enough damage is done to this cellular army for full-blown AIDS to develop. CD4+T lymphocytes are so called because they express a receptor protein termed CD4; this is necessary for T-cell function, but has also been co-opted by HIV and SIV to gain entry to the cells. Despite the apparent low frequency of T-cell infection in chronic infection, seminal studies in SIV-infected monkeys-subsequently confirmed in HIV-infected humans5, 6 - revealed a rapid and widespread depletion of CD4+ T cells in the gut during the first few weeks of infection.

Principles to guide design of an effective vaccine against HIV are greatly needed, particularly to protect women in the pandemic’s epicenter in Africa. We have been seeking these principles by identifying correlates of the robust protection associated with SIVmac239Δnef vaccination in the SIV-rhesus macaque animal model of HIV-1 transmission to women. We identified one correlate of SIVmac239Δnef protection against vaginal challenge as a resident mucosal system for SIV-gp41 trimer Ab production and neonatal FcR-mediated concentration of these Abs on the path of virus entry to inhibit establishment of infected founder populations at the portal of entry. In this study, we identify blocking CD4+ T cell recruitment to thereby inhibit local expansion of infected founder populations as a second correlate of protection. Virus-specific immune complex interactions with the inhibitory FcγRIIb receptor in the epithelium lining the cervix initiate expression of genes that block recruitment of target cells to fuel local expansion. Immune complex–FcγRIIb receptor interactions at mucosal frontlines to dampen the innate immune response to vaginal challenge could be a potentially general mechanism for the mucosal immune system to sense and modulate the response to a previously encountered pathogen. Designing vaccines to provide protection without eliciting these transmission-promoting innate responses could contribute to developing an effective HIV-1 vaccine. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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-l-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lyric assays as well as by gamma interferon (IFN-γ) Elispot assays on cryopre-served 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 lyric 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-γ 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. [ABSTRACT FROM AUTHOR]