Microbiota-Dependent Sequelae of Acute Infection Compromise Tissue-Specific Immunity.

Infections have been proposed as initiating factors for inflammatory disorders; however, identifying associations between defined infectious agents and the initiation of chronic disease has remained elusive. Here, we report that a single acute infection can have dramatic and long-term consequences for tissue-specific immunity. Following clearance of Yersinia pseudotuberculosis, sustained inflammation and associated lymphatic leakage in the mesenteric adipose tissue deviates migratory dendritic cells to the adipose compartment, thereby preventing their accumulation in the mesenteric lymph node. As a consequence, canonical mucosal immune functions, including tolerance and protective immunity, are persistently compromised. Post-resolution of infection, signals derived from the microbiota maintain inflammatory mesentery remodeling and consequently, transient ablation of the microbiota restores mucosal immunity. Our results indicate that persistent disruption of communication between tissues and the immune system following clearance of an acute infection represents an inflection point beyond which tissue homeostasis and immunity is compromised for the long-term. VIDEO ABSTRACT.

SIV infection and ARV treatment reshape the transcriptional and epigenetic profile of naïve and memory T cells in vivo.

Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia.IMPORTANCEChronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences.

Correction: Mitigation of endemic GI-tract pathogen-mediated inflammation through development of multimodal treatment regimen and its impact on SIV acquisition in rhesus macaques.

[This corrects the article DOI: 10.1371/journal.ppat.1009565.].

2-Hydroxypropyl-ß-Cyclodextrin Treatment Induces Modest Immune Activation in Healthy Rhesus Macaques.

Experimental simian immunodeficiency virus (SIV) infection of Asian macaques is an excellent model for HIV disease progression and therapeutic development. Recent coformulations of nucleoside analogs and an integrase inhibitor have been used for parenteral antiretroviral (ARV) administration in SIV-infected macaques, successfully resulting in undetectable plasma SIV RNA. In a cohort of SIVmac239-infected macaques, we recently observed that administration of coformulated ARVs resulted in an unexpected increase in plasma levels of soluble CD14 (sCD14), associated with stimulation of myeloid cells. We hypothesized that the coformulation solubilizing agent Kleptose (2-hydroxypropyl-ß-cyclodextrin [HPßCD]) may induce inflammation with myeloid cell activation and the release of sCD14. Herein, we stimulated peripheral blood mononuclear cells (PBMCs) from healthy macaques with HPßCD from different commercial sources and evaluated inflammatory cytokine production in vitro. Treatment of PBMCs resulted in increased sCD14 release and myeloid cell interleukin-1ß (IL-1ß) production-with stimulation varying significantly by HPßCD source-and destabilized lymphocyte CCR5 surface expression. We further treated healthy macaques with Kleptose alone. In vivo, we observed modestly increased myeloid cell activation in response to Kleptose treatment without significant perturbation of the immunological transcriptome or epigenome. Our results demonstrate a need for vehicle-only controls and highlight immunological perturbations that can occur when using HPßCD in pharmaceutical coformulations. IMPORTANCE SIV infection of nonhuman primates is the principal model system for assessing HIV disease progression and therapeutic development. HPßCD has recently been incorporated as a solubilizing agent in coformulations of ARVs in SIV-infected nonhuman primates. Although HPßCD has historically been considered inert, recent findings suggest that HPßCD may contribute to inflammation. Herein, we investigate the contribution of HPßCD to healthy macaque inflammation in vitro and in vivo. We observe that HPßCD causes an induction of sCD14 and IL-1ß from myeloid cells in vitro and demonstrate that HPßCD stimulatory capacity varies by commercial source. In vivo, we observe modest myeloid cell activation in blood and bronchoalveolar lavage specimens absent systemic immune activation. From our findings, it is unclear whether HPßCD stimulation may improve or diminish immune reconstitution in ARV-treated lentiviral infections. Our results demonstrate a need for vehicle-only controls and highlight immunological perturbations that can occur when using HPßCD in pharmaceutical coformulations.

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates.

Antigen-specific CD8+ T cells play a key role in the host's antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRß gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.

Mitigation of endemic GI-tract pathogen-mediated inflammation through development of multimodal treatment regimen and its impact on SIV acquisition in rhesus macaques.

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal's improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model.

Tissue myeloid cells in SIV-infected primates acquire viral DNA through phagocytosis of infected T cells.

The viral accessory protein Vpx, expressed by certain simian and human immunodeficiency viruses (SIVs and HIVs), is thought to improve viral infectivity of myeloid cells. We infected 35 Asian macaques and African green monkeys with viruses that do or do not express Vpx and examined viral targeting of cells in vivo. While lack of Vpx expression affected viral dynamics in vivo, with decreased viral loads and infection of CD4⁺ T cells, Vpx expression had no detectable effect on infectivity of myeloid cells. Moreover, viral DNA was observed only within myeloid cells in tissues not massively depleted of CD4⁺ T cells. Myeloid cells containing viral DNA also showed evidence of T cell phagocytosis in vivo, suggesting that their viral DNA may be attributed to phagocytosis of SIV-infected T cells. These data suggest that myeloid cells are not a major source of SIV in vivo, irrespective of Vpx expression.

Microbiome Studies in Non-human Primates.

PURPOSE OF REVIEW: Observations of differing bacterial, intestinal microbiomes in people living with HIV have propelled interest in contributions of the microbiome to HIV disease. Non-human primate (NHP) models of HIV infection provide a controlled setting for assessing contributions of the microbiome by standardizing environmental confounders. We provide an overview of the findings of microbiome contributions to aspects of HIV disease derived from these animal models. RECENT FINDINGS: Observations of differing bacterial, intestinal microbiomes are inconsistently observed in the NHP model following SIV infection. Differences in lentiviral susceptibility and vaccine efficacy have been attributed to variations in the intestinal microbiome; however, by-and-large, these differences have not been experimentally assessed. Although compelling associations exist, clearly defined contributions of the microbiome to HIV and SIV disease are lacking. The empirical use of comprehensive multi-omics assessments and longitudinal and interventional study designs in NHP models is necessary to define this contribution more clearly.

Simian Immunodeficiency Virus Infects Functionally Polarized Memory CD4 T Cells Equivalently In Vivo.

Among the numerous immunological abnormalities observed in chronically human immunodeficiency virus (HIV)-infected individuals, perturbations in memory CD4 T cells are thought to contribute specifically to disease pathogenesis. Among these, functional imbalances in the frequencies of T regulatory cells (Tregs) and interleukin 17 (IL-17)/IL-22-producing Th cells (Th17/Th22) from mucosal sites and T follicular helper (Tfh) cells in lymph nodes are thought to facilitate specific aspects of disease pathogenesis. However, while preferential infection of Tfh cells is widely thought to create an important viral reservoir in an immunologically privileged site in vivo, whether immunological perturbations among memory CD4 T cell populations are attributable to their relative infectivity by the virus in vivo is unclear. Here we studied peripheral blood and lymphoid tissues from antiretroviral (ARV)-treated and ARV-naive Asian macaques and isolated functionally defined populations of memory CD4 T cells. We then assessed the degree to which these populations were infected by simian immunodeficiency virus (SIV) in vivo, to determine whether particular functionally identified populations of memory CD4 T cells were preferentially infected by the virus. We found that SIV did not preferentially infect Th17 cells, compared to Th1 cells, Th2 cells, or Tregs. Moreover, Th17 cells contributed proportionately to the total pool of infected cells. Taken together, our data suggest that, although Tfh cells are more prone to harbor viral DNA, other functionally polarized cells are equally infected by the virus in vivo and Th17 cells are not preferentially infected.IMPORTANCE Functional perturbations of memory CD4 T cells have been suggested to underlie important aspects of HIV disease progression. However, the mechanisms underlying these perturbations remain unclear. Using a nonhuman primate model of HIV, we show that SIV infects functionally defined populations of memory CD4 T cells equally in different anatomic sites. Thus, preferential infection by the virus is unlikely to cause functional perturbations.

Antiretroviral Therapy Administration in Healthy Rhesus Macaques Is Associated with Transient Shifts in Intestinal Bacterial Diversity and Modest Immunological Perturbations.

Gastrointestinal (GI) immune system competency is dependent upon interactions with commensal microbiota, which can be influenced by wide-ranging pharmacologic interventions. In simian immunodeficiency virus (SIV)-infected Asian macaque models of human immunodeficiency virus (HIV) infection, we previously noted that initiation of antiretroviral therapy (ART) is associated with a specific imbalance (dysbiosis) of the composition of the intestinal bacteriome. To determine if ART itself might contribute to dysbiosis or immune dysfunction, we treated healthy rhesus macaques with protease, integrase, or reverse transcriptase inhibitors for 1 to 2 or for 5 to 6 weeks and evaluated intestinal immune function and the composition of the fecal bacterial microbiome. We observed that individual antiretrovirals (ARVs) modestly altered intestinal T-cell proinflammatory responses without disturbing total or activated T-cell frequencies. Moreover, we observed transient disruptions in bacterial diversity coupled with perturbations in the relative frequencies of bacterial communities. Shifts in specific bacterial frequencies were not persistent posttreatment, however, with individual taxa showing only isolated associations with T-cell proinflammatory responses. Our findings suggest that intestinal bacterial instability and modest immunological alterations can result from ART itself. These data could lead to therapeutic interventions which stabilize the microbiome in individuals prescribed ART.IMPORTANCE Dysbiosis of the fecal microbiome is a common feature observed in ARV-treated people living with HIV. The degree to which HIV infection itself causes this dysbiosis remains unclear. Here, we demonstrate that medications used to treat HIV infection can influence the composition of the GI tract immune responses and its microbiome in the nonhuman primate SIV model.

Cytotoxic T Cell Functions Accumulate When CD4 Is Downregulated by CD4+ T Cells in African Green Monkeys.

African green monkeys (AGMs) are a natural host of SIV that do not develop simian AIDS. Adult AGMs naturally have low numbers of CD4+ T cells and a large population of MHC class II-restricted CD8αα T cells that are generated through CD4 downregulation in CD4+ T cells. In this article, we study the functional profiles and SIV infection status in vivo of CD4+ T cells, CD8αα T cells, and CD8αß T cells in lymph nodes, peripheral blood, and bronchoalveolar lavage fluid of AGMs and rhesus macaques (in which CD4 downregulation is not observed). We show that, although CD8αα T cells in AGMs maintain functions associated with CD4+ T cells (including Th follicular functionality in lymphoid tissues and Th2 responses in bronchoalveolar lavage fluid), they also accumulate functions normally attributed to canonical CD8+ T cells. These hyperfunctional CD8αα T cells are found to circulate peripherally, as well as reside within the lymphoid tissue. Due to their unique combination of CD4 and CD8 T cell effector functions, these CD4- CD8αα T cells are likely able to serve as an immunophenotype capable of Th1, follicular Th, and CTL functionalities, yet they are unable to be infected by SIV. These data demonstrate the ambiguity of CD4/CD8 expression in dictating the functional capacities of T cells and suggest that accumulation of hyperfunctional CD8αα T cells in AGMs may lead to tissue-specific antiviral immune responses in lymphoid follicles that limit SIV replication in this particular anatomical niche.

Macrophages Are Phenotypically and Functionally Diverse across Tissues in Simian Immunodeficiency Virus-Infected and Uninfected Asian Macaques.

UNLABELLED: Macrophages regulate tissue immunity, orchestrating the initiation and resolution of antimicrobial immune responses and repair of damaged tissue architecture. Their dysfunction can, thus, manifest in either pro- and anti-inflammatory responses. Indeed, despite the importance of macrophage function in health and disease, the role of tissue-resident macrophages in human immunodeficiency virus (HIV) disease progression remains incompletely defined. Here, we use flow cytometry to assess the phenotypes and functions of macrophages isolated from the spleens, axillary lymph nodes, colons, jejuna, and livers of healthy and chronically simian immunodeficiency virus (SIV)-infected Asian macaques, the prominent nonhuman primate model for HIV infection. Our data demonstrate that macrophages from healthy animals exhibit considerable phenotypic and functional heterogeneity across tissues and across a variety of stimuli. Further, our analysis reveals changes in the lipopolysaccharide (LPS) responsiveness of macrophages isolated from SIV-infected animals. We anticipate that our findings will inform future research into macrophage-directed immunity across a variety of primate diseases. IMPORTANCE: These findings highlight the functional and phenotypic heterogeneity of tissue macrophages in different anatomic sites and as a result of SIV infection. We believe that our data will lead to novel therapeutic interventions aimed at altering the proinflammatory capacity of tissue macrophages in progressively HIV-infected individuals.

CD4 depletion in SIV-infected macaques results in macrophage and microglia infection with rapid turnover of infected cells.

In rhesus macaques (RMs), experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

Homeostatic cytokines induce CD4 downregulation in African green monkeys independently of antigen exposure to generate simian immunodeficiency virus-resistant CD8αα T cells.

UNLABELLED: African green monkeys (AGMs; genus Chlorocebus) are a natural host of simian immunodeficiency virus (SIVAGM). As they do not develop simian AIDS, there is great interest in understanding how this species has evolved to avoid immunodeficiency. Adult African green monkeys naturally have low numbers of CD4 T cells and a large population of major histocompatibility complex class II-restricted CD8α(dim) T cells that are generated through CD4 downregulation in CD4(+) T cells. Mechanisms that drive this process of CD4 downregulation are unknown. Here, we show that juvenile AGMs accelerate CD4-to-CD8αα conversion upon SIV infection and avoid progression to AIDS. The CD4 downregulation induced by SIV infection is not limited to SIV-specific T cells, and vaccination of an adult AGM who had a negligible number of CD4 T cells demonstrated that CD4 downregulation can occur without antigenic exposure. Finally, we show that the T cell homeostatic cytokines interleukin-2 (IL-2), IL-7, and IL-15 can induce CD4 downregulation in vitro. These data identify a mechanism that allows AGMs to generate a large, diverse population of T cells that perform CD4 T cell functions but are resistant to SIV infection. A better understanding of this mechanism may allow the development of treatments to induce protective CD4 downregulation in humans. IMPORTANCE: Many African primate species are naturally infected with SIV. African green monkeys, one natural host species, avoid simian AIDS by creating a population of T cells that lack CD4, the human immunodeficiency virus/SIV receptor; therefore, they are resistant to infection. However, these T cells maintain properties of CD4(+) T cells even after receptor downregulation and preserve immune function. Here, we show that juvenile AGMs, who have not undergone extensive CD4 downregulation, accelerate this process upon SIV infection. Furthermore, we show that in vivo, CD4 downregulation does not occur exclusively in antigen-experienced T cells. Finally, we show that the cytokines IL-2, IL-7, and IL-15, which induce homeostatic T cell proliferation, lead to CD4 downregulation in vitro; therefore, they can provide signals that lead to antigen-independent CD4 downregulation. These results suggest that if a similar process of CD4 downregulation could be induced in humans, it could provide a cure for AIDS.

Multifunctional double-negative T cells in sooty mangabeys mediate T-helper functions irrespective of SIV infection.

Studying SIV infection of natural host monkey species, such as sooty mangabeys, has provided insights into the immune changes associated with these nonprogressive infections. Mangabeys maintain immune health despite high viremia or the dramatic CD4 T cell depletion that can occur following multitropic SIV infection. Here we evaluate double-negative (DN)(CD3+CD4-CD8-) T cells that are resistant to SIV infection due to a lack of CD4 surface expression, for their potential to fulfill a role as helper T cells. We first determined that DN T cells are polyclonal and predominantly exhibit an effector memory phenotype (CD95+CD62L-). Microarray analysis of TCR (anti-CD3/CD28) stimulated DN T cells indicated that these cells are multifunctional and upregulate genes with marked similarity to CD4 T cells, such as immune genes associated with Th1 (IFNγ), Th2 (IL4, IL5, IL13, CD40L), Th17 (IL17, IL22) and TFH (IL21, ICOS, IL6) function, chemokines such as CXCL9 and CXCL10 and transcription factors known to be actively regulated in CD4 T cells. Multifunctional T-helper cell responses were maintained in DN T cells from uninfected and SIV infected mangabeys and persisted in mangabeys exhibiting SIV mediated CD4 loss. Interestingly, TCR stimulation of DN T cells from SIV infected mangabeys results in a decreased upregulation of IFNγ and increased IL5 and IL13 expression compared to uninfected mangabeys. Evaluation of proliferative capacity of DN T cells in vivo (BrDU labeling) indicated that these cells maintain their ability to proliferate despite SIV infection, and express the homeostatic cytokine receptors CD25 (IL2 receptor) and CD127 (IL7 receptor). This study identifies the potential for a CD4-negative T cell subset that is refractory to SIV infection to perform T-helper functions in mangabeys and suggests that immune therapeutics designed to increase DN T cell function during HIV infection may have beneficial effects for the host immune system.

CD4+ T cells support production of simian immunodeficiency virus Env antibodies that enforce CD4-dependent entry and shape tropism in vivo.

CD4(+) T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4(+) T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4(+) T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV(+)) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV(+) plasma neutralization. However, plasma from SIV-infected CD4(+) T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4(+) T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4(+) target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.

Untangling the role of the microbiome across the stages of HIV disease.

PURPOSE OF REVIEW: The primate microbiome consists of bacteria, eukaryotes, and viruses that dynamically shape and respond to host health and disease. Understanding how the symbiotic relationship between the host and microbiome responds to HIV has implications for therapeutic design. RECENT FINDINGS: Advances in microbiome identification technologies have expanded our ability to identify constituents of the microbiome and to infer their functional capacity. The dual use of these technologies and animal models has allowed interrogation into the role of the microbiome in lentiviral acquisition, vaccine efficacy, and the response to antiretrovirals. Lessons learned from such studies are now being harnessed to design microbiome-based interventions. SUMMARY: Previous studies considering the role of the microbiome in people living with HIV largely described viral acquisition as an intrusion on the host:microbiome interface. Re-framing this view to consider HIV as a novel, albeit unwelcome, component of the microbiome may better inform the research and development of pre and postexposure prophylaxes.

Immunotoxin-mediated depletion of Gag-specific CD8+ T cells undermines natural control of Simian immunodeficiency virus.

Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is confounded by several factors, including reactive CD4+ T cell proliferation, and further provides no specificity information. We circumvented these limitations by selectively depleting CD8+ T cells specific for the Gag epitope CTPYDINQM (CM9) via the administration of immunotoxin-conjugated tetrameric complexes of CM9/Mamu-A*01. Immunotoxin administration effectively depleted circulating but not tissuelocalized CM9-specific CD8+ T cells, akin to the bulk depletion pattern observed with antibodies directed against CD8. However, we found no evidence to indicate that circulating CM9-specific CD8+ T cells suppressed viral replication in Mamu-A*01+ rhesus macaques during acute or chronic progressive infection with a pathogenic strain of SIV. This observation extended to macaques with established infection during and after continuous antiretroviral therapy. In contrast, natural controller macaques experienced dramatic increases in plasma viremia after immunotoxin administration, highlighting the importance of CD8+ T cell-mediated immunity against CM9. Collectively, these data showed that CM9-specific CD8+ T cells were necessary but not sufficient for robust immune control of SIV in a nonhuman primate model and, more generally, validated an approach that could inform the design of next-generation vaccines against HIV-1.

Decreased plasticity of coreceptor use by CD4-independent SIV Envs that emerge in vivo.

BACKGROUND: HIV and SIV generally require CD4 binding prior to coreceptor engagement, but Env can acquire the ability to use CCR5 independently of CD4 under various circumstances. The ability to use CCR5 coupled with low-to-absent CD4 levels is associated with enhanced macrophage infection and increased neutralization sensitivity, but the additional features of these Envs that may affect cell targeting is not known. RESULTS: Here we report that CD4-independent SIV variants that emerged in vivo in a CD4+ T cell-depleted rhesus macaque model display markedly decreased plasticity of co-receptor use. While CD4-dependent Envs can use low levels of macaque CCR5 for efficient entry, CD4-independent variants required high levels of CCR5 even in the presence of CD4. CD4-independent Envs were also more sensitive to the CCR5 antagonist Maraviroc. CD4-dependent variants mediated efficient entry using human CCR5, whereas CD4-independent variants had impaired use of human CCR5. Similarly, CD4-independent Envs used the alternative coreceptors GPR15 and CXCR6 less efficiently than CD4-dependent variants. Env amino acids D470N and E84K that confer the CD4-independent phenotype also regulated entry through low CCR5 levels and GPR15, indicating a common structural basis. Treatment of CD4-dependent Envs with soluble CD4 enhanced entry through CCR5 but reduced entry through GPR15, suggesting that induction of CD4-induced conformational changes by non-cell surface-associated CD4 impairs use of this alternative co-receptor. CONCLUSIONS: CD4 independence is associated with more restricted coreceptor interactions. While the ability to enter target cells through CCR5 independently of CD4 may enable infection of CD4 low-to-negative cells such as macrophages, this phenotype may conversely reduce the potential range of targets such as cells expressing low levels of CCR5, conformational variants of CCR5, or possibly even alternative coreceptors.

Host Genetics and Environment Shape the Composition of the Gastrointestinal Microbiome in Nonhuman Primates.

The bacterial component of the gastrointestinal tract microbiome is comprised of hundreds of species, the majority of which live in symbiosis with the host. The bacterial microbiome is influenced by host diet and disease history, and host genetics may additionally play a role. To understand the degree to which host genetics shapes the gastrointestinal tract microbiome, we studied fecal microbiomes in 4 species of nonhuman primates (NHPs) held in separate facilities but fed the same base diet. These animals include Chlorocebus pygerythrus, Chlorocebus sabaeus, Macaca mulatta, and Macaca nemestrina. We also followed gastrointestinal tract microbiome composition in 20 Macaca mulatta (rhesus macaques [RMs]) as they transitioned from an outdoor to indoor environment and compared 6 Chlorocebus pygerythrus monkeys that made the outdoor to indoor transition to their 9 captive-born offspring. We found that genetics can influence microbiome composition, with animals of different genera (Chlorocebus versus Macaca) having significantly different gastrointestinal (GI) microbiomes despite controlled diets. Animals within the same genera have more similar microbiomes, although still significantly different, and animals within the same species have even more similar compositions that are not significantly different. Significant differences were also not observed between wild-born and captive-born Chlorocebus pygerythrus, while there were significant changes in RMs as they transitioned into captivity. Together, these results suggest that the effects of captivity have a larger impact on the microbiome than other factors we examined within a single NHP species, although host genetics does significantly influence microbiome composition between NHP genera and species. IMPORTANCE Our data point to the degree to which host genetics can influence GI microbiome composition and suggest, within primate species, that individual host genetics is unlikely to significantly alter the microbiome. These data are important for the development of therapeutics aimed at altering the microbiome within populations of genetically disparate members of primate species.