Glucose Metabolism Disorder Induces Spermatogenic Dysfunction in Northern Pig-Tailed Macaques (Macaca leonina) With Long-Term SIVmac239 Infection.

Although spermatogenic dysfunction is widely found in patients with human immunodeficiency virus (HIV), the underlying reasons remain unclear. Thus far, potential hypotheses involving viral reservoirs, testicular inflammation, hormone imbalance, and cachexia show inconsistent correlation with spermatogenic dysfunction. Here, northern pig-tailed macaques (NPMs) exhibited marked spermatogenic dysfunction after long-term infection with simian immunodeficiency virus (SIVmac239), with significant decreases in Johnsen scores, differentiated spermatogonial stem cells, and testicular proliferating cells. The above hypotheses were also evaluated. Results showed no differences between SIV- and SIV+ NPMs, except for an increase in follicle stimulating hormone (FSH) during SIV infection, which had no direct effect on the testes. However, long-term SIVmac239 infection undermined pancreatic islet ß cell function, partly represented by significant reductions in cellular counts and autophagy levels. Pancreatic islet ß cell dysfunction led to glucose metabolism disorder at the whole-body level, which inhibited lactate production by Sertoli cells in testicular tissue. As lactate is the main energy substrate for developing germ cells, its decrease was strongly correlated with spermatogenic dysfunction. Therefore, glucose metabolism disorder appears to be a primary cause of spermatogenic dysfunction in NPMs with long-term SIVmac239 infection.

Chronic binge alcohol and ovariectomy dysregulate omental adipose tissue metaboproteome in simian immunodeficiency virus-infected female macaques.

Effective antiretroviral therapy (ART) has significantly reduced mortality of people living with HIV (PLWH), and the prevalence of at-risk alcohol use is higher among PLWH. Increased survival and aging of PLWH is associated with increased prevalence of metabolic comorbidities especially among menopausal women, and adipose tissue metabolic dysregulation may be a significant contributing factor. We examined the differential effects of chronic binge alcohol (CBA) administration and ovariectomy (OVX) on the omental adipose tissue (OmAT) proteome in a subset of simian immunodeficiency virus (SIV)-infected macaques of a longitudinal parent study. Quantitative discovery-based proteomics identified 1,429 differentially expressed proteins. Ingenuity Pathway Analysis (IPA) was used to calculate z-scores, or activation predictions, for functional pathways and diseases. Results revealed that protein changes associated with functional pathways centered around the "OmAT metaboproteome profile." Based on z-scores, CBA did not affect functional pathways of metabolic disease but dysregulated proteins involved in adenosine monophosphate-activated protein kinase (AMPK) signaling and lipid metabolism. OVX-mediated proteome changes were predicted to promote pathways involved in glucose- and lipid-associated metabolic disease. Proteins involved in apoptosis, necrosis, and reactive oxygen species (ROS) pathways were also predicted to be activated by OVX and these were predicted to be inhibited by CBA. These results provide evidence for the role of ovarian hormone loss in mediating OmAT metaboproteome dysregulation in SIV and suggest that CBA modifies OVX-associated changes. In the context of OVX, CBA administration produced larger metabolic and cellular effects, which we speculate may reflect a protective role of estrogen against CBA-mediated adipose tissue injury in female SIV-infected macaques.

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

Transient Immune Activation in BCG-Vaccinated Infant Rhesus Macaques Is Not Sufficient to Influence Oral Simian Immunodeficiency Virus Infection.

BCG vaccination has been demonstrated to increase levels of activated CD4+ T cells, thus potentially influencing mother-to-child transmission of human immunodeficiency virus (HIV). To assess the risk of BCG vaccination in HIV infection, we randomly assigned newborn rhesus macaques to receive BCG vaccine or remain unvaccinated and then undergo oral simian immunodeficiency virus (SIV) challenges 3 weeks later. We observed elevated levels of activated peripheral CD4+ T cells (ie, HLA-DR+CD38+CCR5+ CD4+ T cells) by week 3 after vaccination. BCG was also associated with an altered immune gene expression profile, as well as with monocyte activation in both peripheral blood and the draining axillary lymph node, indicating significant BCG vaccine-induced immune activation. Despite these effects, BCG vaccination did not increase the rate of SIV oral transmission or disease progression. Our findings therefore identify patterns of T-cell and monocyte activation that occur after BCG vaccination but do not support the hypothesis that BCG vaccination is a risk factor for postnatal HIV transmission or increased pathogenesis in infants.

Gender differences in innate responses and gene expression profiles in memory CD4 T cells are apparent very early during acute simian immunodeficiency virus infection.

Gender differences in Human immunodeficiency virus (HIV) disease progression and comorbidities have been extensively reported. Using the simian immunodeficiency virus (SIV) infected rhesus macaque model, we show that these differences are apparent very early during the course of infection. Though there were no major changes in the proportions of CD4 T cells or its subsets, central memory CD4 T cells from female macaques were found to differentially regulate a significantly larger number of genes at day 4 post-infection (PI) as compared to males. Pathway analysis revealed divergence of both canonical and biological pathways that persisted at day 10 PI. Changes in gene expression profiles were accompanied by a significant increase in plasma levels of pro-inflammatory mediators such as MCP-1/CCL2, I-TAC/CXCL11, and MIF. Though plasma levels of IFNα did not differ between male and female macaques, the expression levels of IFNα subtype-14, 16, IFNß, and IFNω were significantly upregulated in the lymph nodes of female macaques at day 10 PI as compared to male macaques. Our results suggest that the pathogenic sequelae seen during chronic infection may be shaped by gender differences in immune responses induced very early during the course of HIV infection.

Low levels of SIV-specific CD8+ T cells in germinal centers characterizes acute SIV infection.

CD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection.

Simian immunodeficiency virus transiently increases brain temperature in rhesus monkeys: detection with magnetic resonance spectroscopy thermometry.

PURPOSE: To evaluate brain temperature effects of early simian immunodeficiency virus (SIV) infection in rhesus macaques using proton magnetic resonance spectroscopy (MRS) thermometry (MRSt) and to determine whether temperature correlates with brain choline or myo-inositol levels. METHODS: Brain temperature was retrospectively determined in serial MRS scans that had been acquired at baseline and at 2 and 4 weeks post-SIV infection (wpi) in 16 monkeys by calculating the chemical shift difference between N-acetylaspartate (NAA) and water peaks in sequentially acquired water-suppressed and unsuppressed point-resolved spectroscopy (PRESS) spectra. Frontal and parietal cortex, basal ganglia, and white matter spectra were analyzed. RESULTS: At 2 wpi, brain and rectal temperatures increased relative to baseline and normalized at 4 wpi. Brain temperatures correlated with choline levels in several brain areas, but not with myo-inositol levels. CONCLUSION: These data indicate that SIV transiently increases brain temperature soon after infection and that temperature is correlated with transient changes in choline levels. Given that choline levels are associated with brain inflammation in SIV-infected monkeys, our findings suggest that the SIV-induced temperature increase reflects brain inflammation. We conclude that MRSt may be informative in human immunodeficiency virus models and may be useful for assessing effects of treatments that reduce inflammation. This study also illustrates that existing MRS data sets containing unsuppressed water spectra can be used to measure tissue temperature, an important physiological parameter.

An SIV macaque model of SIV and HAND: the need for adjunctive therapies in HIV that target activated monocytes and macrophages.

Non-human primate models of AIDS and neuroAIDS are critical to study HIV infection of the CNS, neuropathology, and immune activation and macrophage accumulation that occurs in HAND. SIV, similar to HIV, infects CD4+ T lymphocytes and monocytes/macrophages. Virus enters the CNS early, and macrophage activation correlates with CNS disease, as well as inflammation outside of the CNS. Antiretroviral in HIV+ humans and SIV+ Rhesus macaques results in non-detectable plasma virus, decreased or non-detectable viral RNA or protein in the CNS. But, viral DNA rebounds following therapy interruption, demonstrating the presence of replication competent virus in the CNS within myeloid cells. In this brief review, we discuss our findings using a Rhesus macaque model of SIV-associated CNS infection and pathology, focusing on monocyte/macrophage activation and the link between CNS and cardiac disease. We conclude with recent studies using adjunctive therapy targeting monocytes/macrophages with ART to prevent or diminish CNS pathology that may be associated with HAND.

An SIV/macaque model targeted to study HIV-associated neurocognitive disorders.

Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs, to promote HIV cure. In addition to contributing to our understanding of the pathogenesis of HAND, the SIV/pigtailed macaque model also provides an excellent opportunity to test innovative approaches to eliminate the latent HIV reservoir in the brain.

Simian Immunodeficiency Virus Infection Increases Blood Ethanol Concentration Duration After Both Acute and Chronic Administration.

Alcohol use disorder (AUD) is a frequent comorbidity among people living with HIV/AIDS (PLWHA). Alcohol consumption is a significant predictor of nonadherence to antiretroviral therapy (ART), as well as worsening immunological and virological indicators among PLWHA. Clinical studies indicate that higher viral loads increase sensitivity to alcohol in PLWHA. The factors that influence alcohol kinetics after HIV infection and initiation of ART are not well understood, limiting the information upon which interventions can be designed to ameliorate the impact of alcohol misuse on this vulnerable patient population. To better understand the relationship between viral load and alcohol kinetics, we measured changes in doses of intragastric ethanol administration to achieve target blood ethanol concentration (BEC) in a rhesus macaque model of chronic binge alcohol (CBA) administration and acute changes following a single acute binge dose of alcohol (ABA) pre- and post-simian immunodeficiency virus (SIV) infection, and following ART initiation. Our results from CBA (14 months)-administered SIV-infected male macaques showed that, following ART initiation, macaques required higher doses of alcohol to achieve a target peak BEC compared with non-ART-treated SIV-infected macaques. In animals given ABA, we found prolonged duration of elevated BEC and decreased elimination rate of alcohol that was not corrected following 7 weeks of ART. These findings suggest that binge drinking associated with AUD could negatively interact with HIV infection and enhance disease progression. These findings further support the need for implementation of behavioral or therapeutic interventions to decrease alcohol consumption to improve the quality of life in PLWHA.

Modeling aging in HIV infection in nonhuman primates to address an emerging challenge of the post-ART era.

The advent of antiretroviral therapy (ART) has dramatically improved both quality and length of life for subjects infected with human immunodeficiency virus (HIV), delaying or preventing progression to acquired immunodeficiency syndrome (AIDS). However, the virus induces aging-related changes to the immune system which confound treatment. Additionally, the normal physiologic events that occur during aging lead to deficiencies in immunity which not only exacerbate HIV pathogenesis but also trigger a variety of comorbidities. Here, the synergistic linkage between aging and HIV infection is examined in regard to the immunological and pathological mechanisms that drive both senescence and disease progression. The use of NHPs to investigate potential therapeutic strategies to control the deleterious consequences of aging with HIV infection is also reviewed.

KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques.

Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFα) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFα upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.

CD4+ Cell infiltration into subcutaneous adipose tissue is not indicative of productively infected cells during acute SHIV infection.

Limited longitudinal data exist on the effect of HIV on adipose tissue (AT). We found an increase in CD4+ cells and detectable SHIV-RNA in AT during acute SHIV infection. SHIV-RNA+ cells were rare, suggesting that AT is unlikely to be a major source of productively infected cells in SHIV infection.

Decreased myoblast differentiation in chronic binge alcohol-administered simian immunodeficiency virus-infected male macaques: role of decreased miR-206.

Skeletal muscle stem cells play a critical role in regeneration of myofibers. We previously demonstrated that chronic binge alcohol (CBA) markedly attenuates myoblast differentiation potential and myogenic gene expression. Muscle-specific microRNAs (miRs) are implicated in regulation of myogenic genes. The aim of this study was to determine whether myoblasts isolated from asymptomatic CBA-administered simian immunodeficiency virus (SIV)-infected macaques treated with antiretroviral therapy (ART) showed similar impairments and, if so, to elucidate potential underlying mechanisms. Myoblasts were isolated from muscle at 11 mo after SIV infection from CBA/SIV macaques and from time-matched sucrose (SUC)-treated SIV-infected (SUC/SIV) animals and age-matched controls. Myoblast differentiation and myogenic gene expression were significantly decreased in myoblasts from SUC/SIV and CBA/SIV animals compared with controls. SIV and CBA decreased muscle-specific miR-206 in plasma and muscle and SIV decreased miR-206 expression in myoblasts, with no statistically significant changes in other muscle-specific miRs. These findings were associated with a significant increase in histone deacetylase 4 (HDAC4) and decrease in myogenic enhancer factor 2C (MEF2C) expression in CBA/SIV muscle. Transfection with miR-206 inhibitor decreased myotube differentiation, increased expression of HDAC4, and decreased MEF2C, suggesting a critical role of miR-206 in myogenesis. Moreover, HDAC4 was confirmed to be a direct miR-206 target. These results support a mechanistic role for decreased miR-206 in suppression of myoblast differentiation resulting from chronic alcohol and SIV infection. The parallel changes in skeletal muscle and circulating levels of miR-206 warrant studies to establish the possible use of plasma miR-206 as an indicator of impaired muscle function.

A significant productive in vivo infection of resting cells with simian immunodeficiency virus in a macaque with AIDS.

Identifying the cells that can be infected with HIV in vivo and thus potentially persist as latent reservoirs is of high priority. Here, we report the major infected cells in a chronically simian immunodeficiency virus (SIV)-infected macaque that developed AIDS and encephalitis. A majority of the infected cells were detected as non-proliferating resting cells. SIV-infected non-proliferating resting cells were found to be playing an important role in viral pathogenesis, persistence, or reservoir formation.

Chronic binge alcohol administration impairs glucose-insulin dynamics and decreases adiponectin in asymptomatic simian immunodeficiency virus-infected macaques.

Alcohol use disorders (AUDs) frequently exist among persons living with HIV/AIDS. Chronic alcohol consumption, HIV infection, and antiretroviral therapy (ART) are independently associated with impairments in glucose-insulin dynamics. Previous studies from our laboratory have shown that chronic binge alcohol (CBA) administration decreases body mass index, attenuates weight gain, and accentuates skeletal muscle wasting at end-stage disease in non-ART-treated simian immunodeficiency virus (SIV)-infected male rhesus macaques. The aim of this study was to investigate whether CBA and ART alone or in combination alter body composition or glucose-insulin dynamics in SIV-infected male rhesus macaques during the asymptomatic phase of SIV infection. Daily CBA or sucrose (SUC) administration was initiated 3 mo before intrarectal SIV inoculation and continued until the study end point at 11 mo post-SIV infection. ART or placebo was initiated 2.5 mo after SIV infection and continued until study end point. Four treatment groups (SUC/SIV ± ART and CBA/SIV ± ART) were studied. CBA/SIV macaques had significantly decreased circulating adiponectin and resistin levels relative to SUC/SIV macaques and reduced disposition index and acute insulin response to glucose, insulin, and C-peptide release during frequently sampled intravenous glucose tolerance test, irrespective of ART status. No statistically significant differences were observed in homeostatic model assessment-insulin resistance values, body weight, total body fat, abdominal fat, or total lean mass or bone health among the four groups. These findings demonstrate CBA-mediated impairments in glucose-insulin dynamics and adipokine profile in asymptomatic SIV-infected macaques, irrespective of ART.

Gut Mucosal Barrier Dysfunction, Microbial Dysbiosis, and Their Role in HIV-1 Disease Progression.

Distinct pathological events occur within the gastrointestinal (GI) tract of Asian macaques with progressive simian immunodeficiency virus (SIV) infection and humans with human immunodeficiency virus type 1 (HIV-1) infection that are critical in shaping disease course. These events include depletion and functional alteration of GI-resident CD4(+) T cells, loss of antigen-presenting cells, loss of innate lymphocytes, and possible alterations to the composition of the gut microbiota. These contribute to structural damage to the GI tract and systemic translocation of GI tract microbial products. These translocated microbial products directly stimulate the immune system, and there is now overwhelming evidence that this drives chronic immune activation in HIV-1 and SIV infection. While combined antiretroviral therapy (cART) in HIV-1-infected subjects generally allows for immune reconstitution in peripheral blood, reconstitution of the GI tract occurs at a much slower pace, and both immunological and structural abnormalities persist in the GI tract. Importantly, studies of large cohorts of individuals have linked suboptimal GI reconstitution to residual inflammation and heightened morbidities in HIV-1-infected cART recipients. As a result, current era treatments aimed at augmenting restoration of the GI tract hold promise in returning cART recipients to full health.

Effects of Morphine on Behavioral Task Performance in SIV-Infected Rhesus Macaques.

The abuse of opiates such as morphine in synergy with HIV infection not only exacerbates neuropathogenesis but significantly impacts behavioral attributes in HIV infected subjects. Thus, the goal of the current study was to characterize behavioral perturbations in rhesus macaques subjected to chronic morphine and SIV infection. Specifically, we assessed three behavioral tasks: motor skill (MS), forelimb force (FFT) and progressive ratio (PR) tasks. After collecting baseline control data (44 weeks) and data during the morphine-only dependency period (26 weeks), a subset of animals were productively infected with neurovirulent strains of SIVmac (R71/E17) for an additional 33 weeks. A general pattern in the results is that behavioral decline occurred with high CSF viral loads but not necessarily with high plasma viral loads. Compared to saline controls, all treated animals showed significant decreases in performance on all three behavioral tasks during the morphine-only dependency period. During the post infection period, only the morphine plus SIV group showed a significant further decline and this only occurred for the MS task. Taken together, these data demonstrate a clear effect of morphine to produce behavioral deficits and also suggest that morphine can act synergistically with SIV/HIV to exacerbate behavioral deficits.

Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brains of Rhesus Macaques with SIV-Associated Encephalitis.

UNLABELLED: The emergence of a distinct subpopulation of human or simian immunodeficiency virus (HIV/SIV) sequences within the brain (compartmentalization) during infection is hypothesized to be linked to AIDS-related central nervous system (CNS) neuropathology. However, the exact evolutionary mechanism responsible for HIV/SIV brain compartmentalization has not been thoroughly investigated. Using extensive viral sampling from several different peripheral tissues and cell types and from three distinct regions within the brain from two well-characterized rhesus macaque models of the neurological complications of HIV infection (neuroAIDS), we have been able to perform in-depth evolutionary analyses that have been unattainable in HIV-infected subjects. The results indicate that, despite multiple introductions of virus into the brain over the course of infection, brain sequence compartmentalization in macaques with SIV-associated CNS neuropathology likely results from late viral entry of virus that has acquired through evolution in the periphery sufficient adaptation for the distinct microenvironment of the CNS. IMPORTANCE: HIV-associated neurocognitive disorders remain prevalent among HIV type 1-infected individuals, whereas our understanding of the critical components of disease pathogenesis, such as virus evolution and adaptation, remains limited. Building upon earlier findings of specific viral subpopulations in the brain, we present novel yet fundamental results concerning the evolutionary patterns driving this phenomenon in two well-characterized animal models of neuroAIDS and provide insight into the timing of entry of virus into the brain and selective pressure associated with viral adaptation to this particular microenvironment. Such knowledge is invaluable for therapeutic strategies designed to slow or even prevent neurocognitive impairment associated with AIDS.