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A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles (“RNAscope”), vDNA+ cells (“DNAscope”) and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. Highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.

Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses., Author summary Human gammaherpesviruses cause malignancies in HIV infected persons; in SIV infected macaques, gammaherpesvirus infections also occur. To understand the potential role of the rhesus gammaherpesviruses, RRV, RFHV and RLCV in cancers occurring in monkeys during SIV and SHIV studies spanning the last 15 years, we developed assays to measure the DNA viral load (VL) of each virus in these tumors and unaffected macaque tissues. We further characterized the tumors using immunohistochemistry and in situ hybridization, and quantified gammaherpesvirus DNA VL in PBMCs collected longitudinally during the original studies. We examined 18 animals with tumors and 32 controls. In all tumors, we detected one or more gammaherpesviruses; generally, one virus was predominant and very abundant compared to surrounding non-tumor tissues. SIV RNA VL in plasma and RLCV VL in PBMCs were independently associated with cancer risk. The longitudinal patterns of gammaherpesviruses VL, from SIV infection to death differed in cases and controls: while RFHV VL increased only slightly in all animals, RLCV VL and RRV VL increased significantly and continued to increase in cases, but flattened in controls. These data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.

Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)–infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

Early after HIV infection there is substantial depletion of CD4+ T cells in the gastrointestinal (GI) tract lamina propria (LP), with associated epithelial barrier damage, leading to microbial translocation and systemic inflammation and immune activation. In this study, we analyzed these early events in the GI tract in a cohort of Thai acute HIV-infected patients and determined the effect of early combination antiretroviral treatment (cART). HIV-uninfected and chronically and acutely HIV-infected patients at different Fiebig stages (I–V) underwent colonic biopsies and then received cART. Immunohistochemistry and quantitative image analysis were performed on cross-sectional and longitudinal colon biopsy specimens (day 0 to week 96) to measure GI tract damage (infiltration of polymorphonuclear cells), inflammation (Mx1, TNF-α), immune activation (Ki-67), and the CD4+ T cell population in the LP. The magnitude of GI tract damage, immune activation, and inflammation was significantly increased, with significantly depleted CD4+ T cells in the LP in all acutely infected groups prior to cART compared with HIV-uninfected control participants. While most patients treated during acute infection resolved GI tract inflammation and immune activation back to baseline levels after 24 weeks of cART, most acutely infected participants did not restore their CD4+ T cells after 96 weeks of cART.

(See the editorial commentary by Michael on pages 875–6.) Persistent immune activation is a hallmark of human immunodeficiency virus type 1 (HIV-1) infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques. In progressive HIV/SIV infections, the level of immune activation has been demonstrated to be a stronger predictor of disease progression than either plasma viral load or peripheral CD4+ T-cell count [1, 2]. Acute infection is associated with the development of intense immune activation that remains abnormally high throughout the course of infection in the absence of antiretroviral treatment (ART). This persistent pathological immune activation is associated with elevated proinflammatory cytokine and coagulation markers that correlate with an increased risk of death among HIV-positive patients [3]. In SIV-infected monkeys and HIV-infected humans, early augmentation of proinflammatory cytokines and chemokines in lymphoid tissues may play a key role in modulating disease progression [4, 5]. Notably, early high expression of both proinflammatory and antiinflammatory molecules in lymphoid tissues has been proposed to contribute to enhanced immune activation, resulting in tissue damage rather than viral control, thus driving disease progression [4, 6]. Dissecting the elements of the acute inflammatory response that lead to chronic immune activation and disease progression may provide insights that can aid the development of specific adjunctive therapies that may limit inflammation-induced tissue damage and thereby improve the prognosis of HIV-infected individuals. The inflammatory response to infections reflects the aggregate effects of various cytokines and other soluble factors produced by multiple cellular populations. Tumor necrosis factor (TNF) has been identified as a key regulator and amplifier of inflammatory responses, with effects on the innate and adaptive immune systems [7–9]. TNF has been proposed to contribute to the overall pathogenesis of HIV/SIV infection, enhancing viral propagation, lymphocyte depletion, and clinical manifestations of disease. High TNF levels have been shown to be associated with all stages of HIV-1 infection, with increased TNF expression in the plasma and lymphoid tissues during acute HIV-1 infection [10, 11]. Sequential plasma samples collected during the eclipse and exponential viral expansion phases of acute HIV-1 infection demonstrated rapid and robust inflammatory responses, including early and sustained increases in TNF, that likely have immunopathological consequences by promoting immune activation, viral replication, and CD4+ T-cell loss [12]. In nonhuman-primate models, elevated TNF expression has been shown in the serum, peripheral blood mononuclear cells, lymph nodes, and brain of infected animals in temporal association with SIV replication during acute infection [13–15]. In contrast, studies of nonprogressive and apathogenic SIV infection in natural hosts of SIV have shown a lack of TNF upregulation throughout the acute and chronic stages of SIV infection [16], suggesting that differences in the host acute inflammatory response may be critically important in pathogenesis and disease progression. Since TNF inhibitors are used to treat a range of inflammatory conditions [17, 18], and given the data suggesting that TNF may play a key role in the acute inflammatory response to HIV/SIV infection, we evaluated the effect of anti-TNF antibody (adalimumab; Humira) treatment during the first 12 weeks of SIV infection in a small, nonhuman-primate study. While treatment did not affect viral replication dynamics or systemic T-cell activation profiles, inhibition of the biological activity of TNF reduced proinflammatory gene expression in lymphoid tissues during acute infection and attenuated antiinflammatory responses to SIV associated with pathological damage of lymphoid tissues [6]. Specifically, TNF blockade attenuated 3 manifestations of inflammation associated with SIV infection: (1) infiltration of CD163+ macrophages and neutrophils into the paracortical T-cell zone, (2) transforming growth factor β (TGF-β) expression in the T-cell zone, and (3) lymphoid tissue fibrosis, which was associated with less CD4+ T-cell loss, demonstrating that modulation of inflammatory responses to HIV/SIV infection may beneficially affect pathogenesis, independent of any direct effects on viral replication.

CD4+ T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronic HIV infection is a hallmark of disease progression. However, the relative contribution of CD4+ T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4+ T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4+ T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4+ lymphocyte-depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8+ T cell- and B cell-mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4+ T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines.

Mucosal damage to the gastrointestinal (GI) tract with resulting microbial translocation is hypothesized to significantly contribute to the heightened and persistent chronic inflammation and immune activation characteristic to HIV infection. Here we employ a non-human primate model of chemically induced colitis in SIV-uninfected rhesus macaques that we developed using dextran sulfate sodium (DSS), to directly test this hypothesis. DSS treatment results in GI barrier damage with associated microbial translocation, inflammation and immune activation. The progression and severity of colitis are longitudinally monitored by a magnetic resonance imaging approach. DSS treatment of SIV-infected African green monkeys, a natural host species for SIV that does not manifest GI tract damage or chronic immune activation during infection, results in colitis with elevated levels of plasma SIV RNA, sCD14, LPS, CRP and mucosal CD4+ T-cell loss. Together these results support the hypothesis that GI tract damage leading to local and systemic microbial translocation, and associated immune activation, are important determinants of AIDS pathogenesis., HIV-1 infection in humans and SIV infection in rhesus macaques are associated with mucosal damage to the gastrointestinal tract, microbial translocation and chronic immune activation. Here the authors develop a non-human primate DSS colitis model that recapitulates these aspects of the disease in uninfected rhesus macaques.

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques ( Macaca nemestrina ) experimentally infected with XMRV. Following intravenous inoculation with >10 10 RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro , human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.