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Alcohol use has negative effects on HIV disease progression through several mechanisms, including transmission, viral replication, host immunity, and treatment efficacy. Research with animal models has explored the effect of alcohol intake on several aspects of simian immunodeficiency virus (SIV) disease progression. Data suggest that the increased SIV levels observed in alcohol-consuming animals may represent an increase in virus production as opposed to a decrease in host defense. Results also suggest that changes in nutritional balance and metabolism, as a possible consequence of a proinflammatory state, together with increased virus production in animals consuming alcohol, accelerate SIV and possibly HIV disease progression. Further studies using the animal model are necessary. [ABSTRACT FROM AUTHOR]

Introduction: People with pre-existing conditions, including metabolic comorbidities, are at greater risk for complications of SARS-CoV-2 infection and expression of machinery required for viral entry into host cells may be a contributing factor. This study tested the hypothesis that high fat, high sucrose diet (HFSD) and alcohol use increase expression of angiotensin converting enzyme 2 (ACE2) receptor and transmembrane serine protease 2 (TMPRSS2) in tissues isolated from simian immunodeficiency virus (SIV) infected macaques, the most clinically relevant model for the study of HIV., Methods: Biospecimens obtained from a longitudinal study of SIV-infected, antiretroviral therapy (ART)-treated female rhesus macaques ( Macaca mulatta ) were used to determine whether HFSD and chronic binge alcohol (CBA) increased ACE2 and TMPRSS2 protein and gene expression. Macaques (n = 10) were assigned to HFSD or standard diet (SD) for 3 months before CBA or vehicle administration. Three months later, macaques were infected with SIV; ART was initiated 2.5 months thereafter. Tissue samples including lung, pancreas, and kidney were collected at study endpoint (12 months post-SIV infection)., Results: Protein expression of ACE2 in the lung, whole pancreas, and pancreatic islets was significantly greater in HFSD- than SD-fed macaques with no significant differences in protein expression of TMPRSS2 or mRNA expression of ACE2 or TMPRSS2. CBA did not significantly alter any measures., Discussion: The increased ACE2 receptor expression observed in lung and pancreas of SIV-infected HFSD-fed female rhesus macaques aligns with reports that diet may increase susceptibility to COVID-19. These data provide direct evidence for a link between dietary quality and cellular adaptations that may increase the risk for SARS-CoV-2 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Delery, Levitt, Amedee, Molina and Simon.)

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Effective antiretroviral therapy (ART) has transitioned HIV to a chronic disease, with more than 50% of people living with HIV (PLWH) being over the age of 50. HIV targets activated CD4 + T cells expressing HIV-specific co-receptors (CCR5 and CXCR4). Previously, we reported that chronic binge alcohol (CBA)-administered male rhesus macaques had a higher percentage of gut CD4 + T cells expressing simian immunodeficiency virus (SIV) co-receptor CXCR4. Evidence also suggests that gonadal hormone loss increased activated peripheral T cells. Further, mitochondrial function is critical for HIV replication and alcohol dysregulates mitochondrial homeostasis. Hence, we tested the hypothesis that CBA and ovariectomy (OVX) increase circulating activated CD4 + T cells expressing SIV co-receptors and dysregulate mitochondrial homeostasis in SIV-infected female rhesus macaques. Results showed that at the study end-point, CBA/SHAM animals had increased peripheral CD4 + T cell SIV co-receptor expression, and a lower CD4 + T cell count compared to CBA/OVX animals. CBA and OVX animals had altered peripheral immune cell gene expression important for maintaining mitochondrial homeostasis. These results provide insights into how at-risk alcohol use could potentially impact viral expression in cellular reservoirs, particularly in SIV-infected ovariectomized rhesus macaques.

Aging people living with HIV (PLWH), especially postmenopausal women may be at higher risk of comorbidities associated with HIV, antiretroviral therapy (ART), hypogonadism, and at-risk alcohol use. Our studies in simian immunodeficiency virus (SIV)-infected male macaques demonstrated that chronic binge alcohol (CBA) reduced acute insulin response to glucose (AIRG), and at-risk alcohol use decreased HOMA-β in PLWH. The objective of this study was to examine the impact of ovariectomy (OVX) on glucose-insulin dynamics and integrity of pancreatic endocrine function in CBA/SIV-infected female macaques. Female macaques were administered CBA (12-15 g/kg/wk) or isovolumetric water (VEH) intragastrically. Three months after initiation of CBA/VEH administration, all macaques were infected with SIV mac251 , and initiated on antiretroviral therapy (ART) 2.5 mo postinfection. After 1 mo of ART, macaques were randomized to OVX or sham surgeries ( n = 7 or 8/group), and euthanized 8 mo post-OVX (study endpoint). Frequently sampled intravenous glucose tolerance tests (FSIVGTT) were performed at selected time points. Pancreatic gene expression and islet morphology were determined at study endpoint. There was a main effect of CBA to decrease AIRG at Pre-SIV and study endpoint. There were no statistically significant OVX effects on AIRG ( P = 0.06). CBA and OVX decreased the expression of pancreatic markers of insulin docking and release. OVX increased endoplasmic stress markers. CBA but not OVX impaired glucose-insulin expression dynamics in SIV-infected female macaques. Both CBA and OVX altered integrity of pancreatic endocrine function. These findings suggest increased vulnerability of PLWH to overt metabolic dysfunction that may be exacerbated by alcohol use and ovarian hormone loss.

Objective: The present study examined interactions between simian immunodeficiency virus (SIV), chronic binge alcohol (CBA), and antiretroviral therapy (ART) on growth factor signaling, neuroinflammatory markers, viral loads (VL), and CD4+ cell counts., Design: Adult male rhesus macaques were administered CBA (13-14 g ethanol (EtOH)/kg per week) or sucrose (SUC) 3 months prior to SIVmac251 infection until the study endpoint. At viral setpoint, a subset of CBA/SIV+ and SUC/SIV+ macaques were randomized to receive daily ART (9-[2-Phosphonyl-methoxypropyly]adenine [PMPA] 20 mg/kg, 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), 30 mg/kg). Frontal cortex (FC) and basal ganglia (BG) were collected for gene and protein expression., Methods: Relationships between brain and plasma VL or CD4+ cell counts were determined using linear regression. Effects of SIV, CBA, and ART on markers of neuroinflammation and brain-derived neurotrophic factor (BDNF) signaling were determined by ANOVA and linear regression., Results: SIV increased FC and BG neuroinflammatory and glial cell gene expression (CX3CR1, B2M), and reduced FC protein kinase B phosphorylation. CBA decreased FC and BG tropomyosin receptor kinase B (TrkB) phosphorylation, and increased full-length TrkB (TrkB-FL) and SLC1A3 expression in FC and BG, respectively. ART suppressed plasma and brain VL, reduced neuroinflammatory gene expression in FC (IBA1, CX3CR1, and GFAP), and BG (CD74 and CD11ß), and did not restore FC or BG BDNF signaling deficits., Conclusions: Results show ART-mediated reduction in VL and neuroinflammatory gene expression, irrespective of CBA administration. ART did not attenuate SIV- and CBA-mediated BDNF signaling deficits, suggesting these deficits, despite effective neuroinflammation suppression, may explain CBA- and SIV-associated neurocognitive deficits. Therapeutics targeting growth factor signaling may be important adjuvants in treating HIV-associated neurocognitive decline., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

On November 15, 2019, the 24th annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite conference during the annual Society for Leukocyte Biology meeting in Boston, Massachusetts. The 2019 meeting focused on alcohol, immunity, and organ damage, and included two plenary sessions. The first session highlighted new research exploring the mechanisms of alcohol-induced inflammation and liver disease, including effects on lipidomics and lipophagy, regulatory T cells, epigenetics, epithelial cells, and age-related changes in the gut. The second session covered alcohol-induced injury of other organs, encompassing diverse areas of research ranging from neurodegeneration, to lung barrier function, to colon carcinogenesis, to effects on viral infection. The discussions also highlighted current laboratory and clinical research used to identify biomarkers of alcohol use and disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 10 4 TCID 50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 10 5  CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 10 4 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite combination antiretroviral therapies making HIV a chronic rather than terminal condition for many people, the prevalence of HIV-associated neurocognitive disorders (HAND) is increasing. This is especially problematic for children living with HIV. Children diagnosed HAND rarely display the hallmark pathology of HIV encephalitis in adults, namely infected macrophages and multinucleated giant cells in the brain. This finding has also been documented in rhesus macaques infected perinatally with simian immunodeficiency virus (SIV). However, the extent and mechanisms of lack of susceptibility to encephalitis in perinatally HIV-infected children remain unclear. In the current study, we compared brains of macaques infected with pathogenic strains of SIV at different ages to determine neuropathology, correlates of neuroinflammation, and potential underlying mechanisms. Encephalitis was not found in the macaques infected within 24 h of birth despite similar high plasma viral load and high monocyte turnover. Macaques developed encephalitis only when they were infected after 4 months of age. Lower numbers of CCR5-positive cells in the brain, combined with a less leaky blood-brain barrier, may be responsible for the decreased virus infection in the brain and consequently the absence of encephalitis in newborn macaques infected with SIV.

Aims: Microarray analysis of hippocampal tissue from chronic binge alcohol (CBA)-administered, simian immunodeficiency virus (SIV)-infected male macaques identified altered immune response and neurogenesis as potential mechanisms underlying cognitive deficits in macaques. This study investigated the differential brain region associations between markers of neuroinflammation and growth factor signaling with microtubule-associated protein 2 (MAP2) expression., Methods: Adult male rhesus macaques were administered CBA (13-14 g EtOH/kg/week, n = 8) or sucrose (SUC, n = 7) beginning 3 months prior to SIV infection and continued until animals reached end-stage disease criteria (3-24 months post infection). Expression of inflammatory cytokines, growth factors, and viral loads were determined in the prefrontal cortex (PFC), caudate (CD), and hippocampus (HP). Brain-derived neurotropic factor (BDNF) expression and phosphorylation of intracellular kinases downstream of BDNF were investigated in the PFC., Results: Our results show reduced MAP2 expression in the PFC of longer-surviving, CBA/SIV macaques. BDNF expression was most closely associated with MAP2 expression in the PFC. In the caudate, significant positive associations were observed between MAP2 and BDNF, time to end-stage and set-point viral load and significant negative associations for CBA. In the hippocampus, positive associations were observed between MAP2 and inflammatory cytokines, and negative associations for brain viral load and CBA., Conclusions: CBA differentially affects growth factor and inflammatory cytokine expression and viral load across brain regions. In the PFC, suppression of growth factor signaling may be an important neuropathological mechanism, while inflammatory processes may play a more important role in the CD and HP., (© The Author(s) 2019. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Short Summary: : Effective combined antiretroviral therapy regimens have extended survival of persons living with HIV (PLWH). Heavy alcohol consumption is common in PLWH. This overview integrates evidence from clinical and preclinical research to identify salient alcohol-related mechanisms and comorbidities contributing to disease pathogenesis and accelerated aging and senescence in PLWH.

Despite optimization of preventative measures for vertical HIV-1 transmission, daily, roughly 400 infants become HIV infected, most of them through breastfeeding. Viral entry has been presumed to occur in the gastrointestinal tract; however, the exact entry site(s) have not been defined. Therefore, we quantified simian immunodeficiency virus (SIV) RNA and DNA in oral, intestinal, and systemic tissues of 15 infant macaques within 48-96 h after oral SIV mac251 exposure. SIV DNA was detected as early as 48 h, whereas SIV RNA was typically detected at later time points (72-96 h). Transmitted founder viruses were identical or very similar to a single genotype in the SIV mac251 challenge stock. SIV RNA and DNA were most frequently found in lymph nodes (LNs) draining the oral cavity and in the ileum. Using in situ hybridization, SIV-infected cells in LNs were exclusively represented by CD3 + T cells. SIV RNA and DNA were also detected in the lungs of 20% of the animals, and 60% of the animals had detectable SIV DNA in the cerebrum. The early detection of viral RNA or DNA in lung and brain tissues emphasizes the need for early treatment of pediatric HIV infection to prevent damage not only to the immune system but also to the respiratory tract and central nervous system.

Persistent HIV reservoirs and the absolute quantification of viral RNA copies in tissues have become a prominent focus of multiple areas ofHIV/SIV research. Absolute quantification of viral RNA via reverse transcription, quantitative PCR (RT-qPCR) necessitates the use of an appropriate RNA reference gene whose expression is unaffected by both experimental and confounding conditions. In this study, we demonstrate the utility of ribosomal protein S13 mRNA (RPS13) as a stable, medium abundance reference gene for RT-qPCR normalization of HIV/SIV RNA copy number. We developed a RPS13 RNA standard assay utilizing an in vitro RNA transcript for normalization of absolute SIV RNA quantities in tissues reservoirs. The RT-qPCR assay showed a high degree of repeatability and reproducibility across RNA levels appropriate for absolute SIV quantification. In assessing the utility of RPS13 as a reference gene, limited variation in the absolute, inter-tissue quantities of RPS13 mRNA was observed within multiple tissue samples obtained from rhesus macaques (average CV=2.86%). We demonstrate rhesus macaque RPS13 mRNA expression is not affected by alcohol administration, SIV infection, or antiviral therapy (PMPA/FTC). Additionally, assay functionality was validated for normalization of SIV copy number using cellular RNA prepared from samples of variable RNA integrity. RPS13 is a suitable reference gene for normalization of absolute SIV RNA quantities in tissues and is most appropriate for intra-tissue or similar tissue type comparisons of SIV copy number., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Despite significant progress in reducing peripartum mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor, and breast milk exposure to HIV accounts for up to 44% of MTCT. As abstinence from breastfeeding is not recommended, alternative means are needed to prevent MTCT of HIV. We have previously shown that oral vaccination at birth with live attenuated Mycobacterium tuberculosis strains expressing simian immunodeficiency virus (SIV) genes safely induces persistent SIV-specific cellular and humoral immune responses both systemically and at the oral and intestinal mucosa. Here, we tested the ability of oral M. tuberculosis vaccine strains expressing SIV Env and Gag proteins, followed by systemic heterologous (MVA-SIV Env/Gag/Pol) boosting, to protect neonatal macaques against oral SIV challenge. While vaccination did not protect infant macaques against oral SIV acquisition, a subset of immunized animals had significantly lower peak viremia which inversely correlated with prechallenge SIV Env-specific salivary and intestinal IgA responses and higher-avidity SIV Env-specific IgG in plasma. These controller animals also maintained CD4(+) T cell populations better and showed reduced tissue pathology compared to noncontroller animals. We show that infants vaccinated at birth can develop vaccine-induced SIV-specific IgA and IgG antibodies and cellular immune responses within weeks of life. Our data further suggest that affinity maturation of vaccine-induced plasma antibodies and induction of mucosal IgA responses at potential SIV entry sites are associated with better control of viral replication, thereby likely reducing SIV morbidity., Importance: Despite significant progress in reducing peripartum MTCT of HIV with ART, continued access to ART throughout the breastfeeding period is still a limiting factor. Breast milk exposure to HIV accounts for up to 44% of MTCT. Alternative measures, in addition to ART, are needed to achieve the goal of an AIDS-free generation. Pediatric HIV vaccines constitute a core component of such efforts. The results of our pediatric vaccine study highlight the potential importance of vaccine-elicited mucosal Env-specific IgA responses in combination with high-avidity systemic Env-specific IgG in protection against oral SIV transmission and control of viral replication in infant macaques. The induction of potent mucosal IgA antibodies by our vaccine is remarkable considering the age-dependent development of mucosal IgA responses postbirth. A deeper understanding of postnatal immune development may inform the design of improved vaccine strategies to enhance systemic and mucosal SIV/HIV antibody responses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIVBaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.

Problem: HIV infection among women is frequently modeled in female rhesus macaques. Longitudinal studies on genital compartment and hormonal factors that can influence susceptibility to SIV infection are lacking in this animal model., Method of Study: Genital specimens and menstruation of indoor-housed female rhesus macaques were analyzed prior to and after SIV infection., Results: Median menstrual cycle length averaged 27 days, although highly variable cycle lengths and frequent periods of amenorrhea were observed during summer months. The vaginal microbiota, characterized by adapted Nugent scoring, showed predominance of small Gram-variable rods and Gram-positive cocci. Highly variable vaginal cytokine levels were observed pre- and post-SIV infection. Vaginal viral loads correlated with plasma viral loads, but were not associated with progesterone levels., Conclusion: These results provide an integrated characterization of important factors in the vaginal microenvironment that are relevant to the experimental design of HIV prevention and transmission studies in female rhesus macaques., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HIV-associated mortality has been significantly reduced with antiretroviral therapy (ART), and HIV infection has become a chronic disease that frequently coexists with many disorders, including substance abuse (Azar et al. Drug Alcohol Depend 112:178-193, 2010; Phillips et al. J Gen Int Med 16:165, 2001). Alcohol and drugs of abuse may modify host-pathogen interactions at various levels including behavioral, metabolic, and immune consequences of HIV infection, as well as the ability of the virus to integrate into the genome and replicate in host cells. Identifying mechanisms responsible for these interactions is complicated by many factors, such as the tissue specific responses to viral infection, multiple cellular mechanisms involved in inflammatory responses, neuroendocrine and localized responses to infection, and kinetics of viral replication. An integrated physiological analysis of the biomedical consequences of chronic alcohol and drug use or abuse on disease progression is possible using rhesus macaques infected with simian immunodeficiency virus (SIV), a relevant model of HIV infection. This review will provide an overview of the data gathered using this model to show that chronic administration of two of the most commonly abused substances, alcohol and cannabinoids (Δ(9)-Tetrahydrocannabinol; THC), affect host-pathogen interactions.

HIV disease and alcohol independently influence the human immune system, so it stands to reason that, together, their influence may be additive. Here, we review the evidence that alcohol can exacerbate HIV's influence on the immune system, thereby affecting disease progression and transmission. We focus particularly on alcohol's effect on the mucosal immune system in the tissues of the gastrointestinal tract, the genital tract and the lungs, all of which play a role in transmission and progression of HIV disease.

Persons living with HIV/AIDS (PLWHA) frequently use cannabinoids, either recreationally by smoking marijuana or therapeutically (delta-9-tetrahydrocannabinol; Δ(9)-THC dronabinol). Previously, we demonstrated that chronic Δ(9)-THC administration decreases early mortality in male simian immunodeficiency virus (SIV)-infected macaques. In this study, we sought to examine whether similar protective effects resulted from chronic cannabinoid administration in SIV-infected female rhesus macaques. Clinical and viral parameters were evaluated in eight female rhesus macaques that received either Δ(9)-THC (0.18-0.32 mg/kg, intramuscularly, twice daily) or vehicle (VEH) starting 28 days prior to intravenous inoculation with SIVmac251. SIV disease progression was assessed by changes in body weight, mortality, viral levels in plasma and mucosal sites, and lymphocyte subsets. In contrast to our results in male animals, chronic Δ(9)-THC did not protect SIV-infected female rhesus macaques from early mortality. Markers of SIV disease, including viral load and CD4(+)/CD8(+) ratio, were not altered by Δ(9)-THC compared to control females; however, females that received chronic Δ(9)-THC did not gain as much weight as control animals. In addition, Δ(9)-THC administration increased total CXCR4 expression in both peripheral and duodenal CD4(+) and CD8(+) T lymphocytes prior to SIV inoculation. Although protection from early mortality was not evident, chronic Δ(9)-THC did not affect clinical markers of SIV disease progression. The contrasting effects of chronic Δ(9)-THC in males versus females remain to be explained, but highlight the need for further studies to explore the sex-dependent effects of Δ(9)-THC and other cannabinoids on the HIV disease course and their implications for virus transmission.

Background: Alcohol use disorders (AUDs) are a frequent comorbidity in a large percentage of people living with HIV/AIDS (PLWHA). PLWHA with comorbid AUDs are consistently found to perform poorly at most levels of the HIV treatment cascade, resulting in a higher likelihood of virologic nonsuppression. This has been partly attributed to lower rates of persistence with and adherence to antiretroviral therapies (ART). Focus groups of in-care PLWHA identify the need to suspend ART on drinking days because of the potential for toxicity and/or lack of therapeutic effectiveness. The aim of this study was to examine whether chronic binge alcohol (CBA) consumption decreases the effectiveness of uninterrupted ART, specifically that of nucleoside reverse-transcriptase inhibitors (NRTI) tenofovir and emtricitabine in suppressing viral replication, or results in drug toxicity in simian immunodeficiency virus (SIV)-infected rhesus macaques., Methods: Daily CBA or isocaloric sucrose (SUC) administration was initiated 3 months prior to intrarectal SIVmac251 inoculation and continued throughout the study period. ART was initiated 2.5 months after SIV infection and continued through the study period., Results: CBA administration did not prevent or delay the ART-mediated reduction in viral load. Following ART, circulating levels of total protein and creatinine were significantly higher than baseline values in both SUC- and CBA-treated animals, but still within a normal range. No evidence of ART toxicity was observed in either CBA- or SUC-administered macaques., Conclusions: These findings indicate that CBA does not attenuate effectiveness of NRTI suppression of viral load, nor does it appear to interact with NRTI to produce toxicity during the initial 2 months of treatment. We conclude that while efforts to reduce AUD in PLWHA should be a priority, counseling on the importance of adherence to ART even on drinking days should also be promoted., (Copyright © 2014 by the Research Society on Alcoholism.)

Alcohol abuse is a widespread problem among those at risk for and living with HIV and can impact transmission and disease progression. In this study we sought to use the simian immunodeficiency virus (SIV)-macaque model to evaluate the immunological and virological changes in the genital microenvironment of females exposed to chronic alcohol. Female rhesus macaques were treated with alcohol (n=6) or isocaloric sucrose (n=6) for 3 months and then inoculated with SIVmac251. To assess the effects of chronic alcohol on SIV disease and the genital microenvironment, we quantified plasma and genital SIV levels, measured inflammatory cells in genital fluids, and characterized microbial flora by gram stains over 10 weeks post-SIV infection. Following 3 months of alcohol/sucrose treatment, significant differences were observed in the vaginal microenvironment of alcohol-treated animals as compared to controls. Microbial flora of alcohol-treated animals had decreased levels of lactobacillus morphotypes and increased levels of gram-positive cocci relative to sucrose controls. Alcohol-treated animals were also more likely to have white blood cells in vaginal fluids prior to SIV inoculation, which persisted through viral set point. Similar levels of cell-free SIV were observed in plasma and vaginal fluids of both groups, but alcohol-treated animals had a higher incidence and levels of cell-associated SIV shed in vaginal secretions. Chronic alcohol treatment negatively impacts the genital microenvironment prior to and over the course of SIV infection and may increase the risk of genital virus shedding and transmission.

Our studies have demonstrated that chronic Δ(9)-tetrahydrocannabinol (THC) administration results in a generalized attenuation of viral load and tissue inflammation in simian immunodeficiency virus (SIV)-infected male rhesus macaques. Gut-associated lymphoid tissue is an important site for HIV replication and inflammation that can impact disease progression. We used a systems approach to examine the duodenal immune environment in 4- to 6-year-old male rhesus monkeys inoculated intravenously with SIVMAC251 after 17 months of chronic THC administration (0.18-0.32 mg/kg, intramuscularly, twice daily). Duodenal tissue samples excised from chronic THC- (N=4) and vehicle (VEH)-treated (N=4) subjects at ∼5 months postinoculation showed lower viral load, increased duodenal integrin beta 7(+)(β7) CD4(+) and CD8(+) central memory T cells, and a significant preferential increase in Th2 cytokine expression. Gene array analysis identified six genes that were differentially expressed in intestinal samples of the THC/SIV animals when compared to those differentially expressed between VEH/SIV and uninfected controls. These genes were identified as having significant participation in (1) apoptosis, (2) cell survival, proliferation, and morphogenesis, and (3) energy and substrate metabolic processes. Additional analysis comparing the duodenal gene expression in THC/SIV vs. VEH/SIV animals identified 93 differentially expressed genes that participate in processes involved in muscle contraction, protein folding, cytoskeleton remodeling, cell adhesion, and cell signaling. Immunohistochemical staining showed attenuated apoptosis in epithelial crypt cells of THC/SIV subjects. Our results indicate that chronic THC administration modulated duodenal T cell populations, favored a pro-Th2 cytokine balance, and decreased intestinal apoptosis. These findings reveal novel mechanisms that may potentially contribute to cannabinoid-mediated disease modulation.

The populations at risk for HIV infection, as well as those living with HIV, overlap with populations that engage in heavy alcohol consumption. Alcohol use has been associated with high-risk sexual behavior and an increased likelihood of acquiring HIV, as well as poor outcome measures of disease such as increased viral loads and declines in CD4+ T lymphocytes among those living with HIV-infections. It is difficult to discern the biological mechanisms by which alcohol use affects the virus:host interaction in human populations due to the numerous variables introduced by human behavior. The rhesus macaque infected with simian immunodeficiency virus has served as an invaluable model for understanding HIV disease and transmission, and thus, provides an ideal model to evaluate the effects of chronic alcohol use on viral infection and disease progression in a controlled environment. In this review, we describe the different macaque models of chronic alcohol consumption and summarize the studies conducted with SIV and alcohol. Collectively, they have shown that chronic alcohol consumption results in higher levels of plasma virus and alterations in immune cell populations that potentiate SIV replication. They also demonstrate a significant impact of chronic alcohol use on SIV-disease progression and survival. These studies highlight the utility of the rhesus macaque in deciphering the biological effects of alcohol on HIV disease. Future studies with this well-established model will address the biological influence of alcohol use on susceptibility to HIV, as well as the efficacy of anti-retroviral therapy.

Background: Simian immunodeficiency virus (SIV) infection in macaques chronically receiving ethanol results in significantly higher plasma viral loads and more rapid progression to end-stage disease. We thus hypothesized that the increased plasma viral load in ethanol-treated, SIV-infected macaques would negatively correlate with antigen-specific immune responses., Methods: Rhesus macaques were administered ethanol or sucrose (n = 12 per group) by indwelling gastric catheters for 3 months and then intravenously infected with SIVMAC251. Peripheral blood T- and B-cell immunophenotyping and quantification were performed. Plasma was examined for viremia, levels of SIVEnv-specific binding, and neutralizing antibodies. Virus-specific interferon γ and tumor necrosis factor α cytokine responses to SIV-Nef, Gag, or Env peptide pools were measured in peripheral blood CD8 T cells., Results: Macaques receiving ethanol had both higher plasma viremia and virus-specific cellular immune responses compared with the sucrose-treated group. The emergence of virus-specific cytokine responses temporally correlated with the decline in mean plasma viral load after 14 days postinfection in all SIV-infected animals. However, neither the breadth and specificity nor the magnitude of virus-specific CD8 T-cell responses correlated with early postpeak reductions in plasma viral loads. In fact, increased cytokine responses against Gag, gp120, and gp41 positively correlated with plasma viremia. Levels of SIV envelope-specific immunoglobulin G and neutralizing antibodies were similar over the disease course in both groups of macaques., Conclusions: Persistently higher antigen-specific cytokine responses in animals receiving ethanol are likely an effect of the higher viral loads and antigen persistence, rather than a cause of the increased viremia.

Background: The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru., Methods: Between 2002-5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7·105 RNA/ml (range: 2.1·10(3) - 1.2·10(6)), and a median CD4-count of 232 cells/μL (range: 1-1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots., Results: During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure., Conclusions: Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/- NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure.

Among the now pandemic sexually transmitted infections (STIs), Chlamydia trachomatis (C. trachomatis) is the predominant bacterial pathogen and human immunodeficiency virus type 1 (HIV-1) is the most lethal of the viral pathogens. The female genital tract is the primary site for heterosexual transmission of both C. trachomatis and HIV-1. Infection with C. trachomatis, and with a variety of other STIs, increases the risk for transmission of HIV-1, although the mechanisms for this finding remain unclear. We have used in vitro modeling to assess the mechanisms by which infection with genital C. trachomatis serovars might increase the transmission of HIV-1 across the female genital tract. C. trachomatis infection of an immortalized endocervical epithelial cell line (A2EN) increases the cell surface expression of the HIV-1 alternative primary receptor, galactosyl ceramide (GalCer), and of the HIV-1 co-receptors, CXCR4 and CCR5. C. trachomatis infection also increases the binding of HIV-1 to A2EN cells, and, subsequently, increases levels of virus in co-cultures of HIV-exposed A2EN and susceptible MT4-R5 T cells. Finally, in vivo endocervical cell sampling reveals a dramatic increase in the number of CD4+, CXCR4 and/or CCR5 positive T cell targets in the endocervix of C. trachomatis positive women when compared to those who are C. trachomatis negative. This combination of in vitro and in vivo results suggests several mechanisms for increased transmission of HIV-1 across the endocervices of C. trachomatis-infected women.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.

Perinatal HIV transmission occurs in utero or intrapartum. The mechanisms and timing of transmission are not clearly understood. To compare the genetic sequences of the V3 envelope region of infant's plasma HIV to that of the mother's plasma, peripheral blood mononuclear cells (PBMC) and vaginal secretions, and correlate with timing of transmission. All 3 infants had a positive HIV PCR in the first days of life, thus classified as in utero infections. In the first mother-infant pair, two different variants were present in the infant, one correlating with maternal PBMC virus and highly homologous to virus from vaginal secretions and the other identical to sequences in maternal plasma. In the second pair, the infant plasma virus was similar to that of maternal PBMC. In the third pair, the cord blood and infant plasma virus were highly similar to maternal vaginal virus. The presence of more than one HIV variant from the maternal blood and from the vaginal compartment in the cord blood of infants presumably infected in utero could point to more than one episode of transmission or, alternatively, to transmission of PBMC virus.

Although Δ⁹-THC has been approved to treat anorexia and weight loss associated with AIDS, it may also reduce well-being by disrupting complex behavioral processes or enhancing HIV replication. To investigate these possibilities, four groups of male rhesus macaques were trained to respond under an operant acquisition and performance procedure, and administered vehicle or Δ⁹-THC before and after inoculation with simian immunodeficiency virus (SIV(mac251), 100 TCID₅₀/ml, i.v.). Prior to chronic Δ⁹-THC and SIV inoculation, 0.032-0.32 mg/kg of Δ⁹-THC produced dose-dependent rate-decreasing effects and small, sporadic error-increasing effects in the acquisition and performance components in each subject. Following 28 days of chronic Δ⁹-THC (0.32 mg/kg, i.m.) or vehicle twice daily, delta-9-THC-treated subjects developed tolerance to the rate-decreasing effects, and this tolerance was maintained during the initial 7-12 months irrespective of SIV infection (i.e., +THC/-SIV, +THC/+SIV). Full necropsy was performed on all SIV subjects an average of 329 days post-SIV inoculation, with postmortem histopathology suggestive of a reduced frequency of CNS pathology as well as opportunistic infections in delta-9-THC-treated subjects. Chronic Δ⁹-THC also significantly reduced CB-1 and CB-2 receptor levels in the hippocampus, attenuated the expression of a proinflammatory cytokine (MCP-1), and did not increase viral load in plasma, cerebrospinal fluid, or brain tissue compared to vehicle-treated subjects with SIV. Together, these data indicate that chronic Δ⁹-THC produces tolerance to its behaviorally disruptive effects on complex tasks while not adversely affecting viral load or other markers of disease progression during the early stages of infection.

Background: Identification of factors associated with the presence of human immunodeficiency virus (HIV) in female genital secretions is critical for intervention strategies targeting transmission and eliminating replication of genital virus. We sought to monitor the prevalence of genital HIV shedding in antiretroviral therapy-adherent women over time and to assess changes in the genital microenvironment., Methods: Levels of cell-free HIV (HIV RNA) and HIV-infected cells (HIV DNA) were monitored in peripheral blood samples and cervical and vaginal fluid samples at monthly intervals in 11 women for 1 year. Genital tract infections and fluctuations in cervical and vaginal white blood cell counts were also evaluated at each study visit., Results: Plasma HIV was undetectable at the majority of study visits; when detected, it was only at low levels. Throughout the study, genital HIV RNA and DNA were detected in each person. Combined genital HIV (RNA and DNA) was detected at 49.2% of study visits and was associated with an elevated concentration of cervical white blood cell infiltrate (odds ratio, 2.52 [95% confidence interval, 1.01-6.22]; P = .04). Infiltrate was not associated with a clinical disorder or patient-reported symptoms., Conclusions: Despite antiretroviral therapy adherence and clinically suppressed plasma viremia, HIV was intermittently detected in genital secretions and was associated with subclinical inflammation and cells trafficking to the cervical mucosa.

Human immunodeficiency virus (HIV) infections are rarely acquired via an oral route in adults. Previous studies have shown that human whole saliva inhibits HIV infection in vitro, and multiple factors present in human saliva have been shown to contribute to this antiviral activity. Despite the widespread use of simian immunodeficiency virus (SIV)-infected rhesus macaques as models for HIV pathogenesis and transmission, few studies have monitored SIV in the oral cavity of infected rhesus macaques and evaluated the viral inhibitory capacity of macaque saliva. Utilizing a cohort of rhesus macaques infected with SIV(Mac251), we monitored virus levels and genotypic diversity in the saliva throughout the course of the disease; findings were similar to previous observations in HIV-infected humans. An in vitro infectivity assay was utilized to measure inhibition of HIV/SIV infection by normal human and rhesus macaque whole saliva. Both human and macaque saliva were capable of inhibiting HIV and SIV infection. The inhibitory capacity of saliva samples collected from a cohort of animals postinfection with SIV increased over the course of disease, coincident with the development of SIV-specific antibodies in the saliva. These findings suggest that both innate and adaptive factors contribute to inhibition of SIV by whole macaque saliva. This work also demonstrates that SIV-infected rhesus macaques provide a relevant model to examine the innate and adaptive immune responses that inhibit HIV/SIV in the oral cavity.

Background: Quantifying HIV levels in mucosal secretions is essential to study compartmentalized expression of HIV and facilitate development of intervention strategies to prevent disease progression and transmission., Objectives: To develop a sensitive, reliable, and cost-effective technique to quantify HIV from blood and vaginal secretions that is compatible with efficient implementation in clinical research environments., Study Design: A sensitive, reliable, internally-controlled real-time reverse transcriptase (RT) PCR assay, which uses the HIV-1 pol gene as a target (Hpol assay) was developed to quantify HIV levels in plasma and genital secretions, and compared to the widely used Roche Amplicor HIV-1 Monitor assay. In addition, a simplified method of sample collection and processing of genital secretions (self-collection and use of RNAlater with batch processing) was compared to provider collection of samples and immediate processing., Results: The sensitivity and reliability of HIV levels detected by the assay described herein correlate well with measurements from Roche Amplicor HIV-1 Monitor assay for both plasma and vaginal secretions (R(2)=0.9179 and R(2)=0.942, respectively). The Hpol assay reproducibly quantifies a lower limit of 5 HIV-1 RNA copies per reaction, with low-levels of inter-assay and intra-assay variation. Additionally, vaginal viral levels and detection frequency did not differ significantly between the two the collection and processing methods., Conclusions: The methodologies developed here provide sensitive, reliable, and cost-effective quantification of HIV levels in plasma and mucosal secretions, and are compatible with efficient use in clinical research studies.

The world is experiencing a pandemic of influenza that emerged in March 2009, due to a novel strain designated influenza A/H1N1 2009. This strain is closest in molecular sequence to swine influenza viruses, but differs from all previously known influenza by a minimum of 6.1%, and from prior "seasonal" H1N1 by 27.2%, giving it great potential for widespread human infection. While spread into India was delayed for two months by an aggressive interdiction program, since 1 August 2009 most cases in India have been indigenous. H1N1 2009 has differentially struck younger patients who are naïve susceptibles to its antigenic subtype, while sparing those >60 who have crossreactive antibody from prior experience with influenza decades ago and the 1977 "swine flu" vaccine distributed in the United States. It also appears to more severely affect pregnant women. It emanated from a single source in central Mexico, but its precise geographical and circumstantial origins, from either Eurasia or the Americas, remain uncertain. While currently a mild pandemic by the standard of past pandemics, the seriousness of H1N1 2009 especially among children should not be underestimated. There is potential for the virus, which continues to adapt to humans, to change over time into a more severe etiologic agent by any of several foreseeable mutations. Mass acceptance of the novel H1N1 2009 vaccine worldwide will be essential to its control. Having spread globally in a few months, affecting millions of people, it is likely to remain circulating in the human population for a decade or more.

Background: Dual epidemics of the malaria parasite Plasmodium and HIV-1 in sub-Saharan Africa and Asia present a significant risk for co-infection in these overlapping endemic regions. Recent studies of HIV/Plasmodium falciparum co-infection have reported significant interactions of these pathogens, including more rapid CD4+ T cell loss, increased viral load, increased immunosuppression, and increased episodes of clinical malaria. Here, we describe a novel rhesus macaque model for co-infection that supports and expands upon findings in human co-infection studies and can be used to identify interactions between these two pathogens., Methodology/principal Findings: Five rhesus macaques were infected with P. cynomolgi and, following three parasite relapses, with SIV. Compared to macaques infected with SIV alone, co-infected animals had, as a group, decreased survival time and more rapid declines in markers for SIV progression, including peripheral CD4+ T cells and CD4+/CD8+ T cell ratios. The naïve CD4+ T cell pool of the co-infected animals was depleted more rapidly than animals infected with SIV alone. The co-infected animals also failed to generate proliferative responses to parasitemia by CD4+ and CD8+ T cells as well as B cells while also having a less robust anti-parasite and altered anti-SIV antibody response., Conclusions/significance: These data suggest that infection with both SIV and Plasmodium enhances SIV-induced disease progression and impairs the anti-Plasmodium immune response. These data support findings in HIV/Plasmodium co-infection studies. This animal model can be used to further define impacts of lentivirus and Plasmodium co-infection and guide public health and therapeutic interventions.

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at -20 degrees C. DBS were created by spotting 50-microl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at -20 degrees C or at 37 degrees C with approximately 85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at -20 degrees C, and 398 stored at 37 degrees C. A chi-square test showed fewer positive reactions for DBS stored at 37 degrees C (55%) than for those stored at -20 degrees C (78%) (P < 0.0001). Samples stored at -20 degrees C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37 degrees C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37 degrees C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.

HIV expressed in genital secretions provides the inoculum from which transmitting variants are selected, both in sexual transmission and mother-to-infant transmission during partuition. Characterization of HIV levels and genotypes found in vaginal secretions and the impact of anti-retroviral therapy (ART) on this virus can provide valuable insight for the prevention of HIV transmission. Vaginal HIV was evaluated in a cohort of 43 women attending a New Orleans HIV outpatient clinic. Predominant vaginal genotypes were characterized as R5- or X4-like by heteroduplex tracking analyses of the envelope V3 region. Most women (67.4%) shed R5-like genotypes in vaginal secretions which was associated with elevated plasma HIV levels (>or= 10,000 copies HIV-RNA/mL) and absence of ART. Because R5-like genotypes are more frequently associated with transmission, these observations suggest that the majority of women shedding HIV in genital secretions present a transmission risk. The levels of vaginal virus were similar between both groups, but shedding of X4-like genotypes was associated with lower plasma viral loads and the use of ART, suggesting that ART use may impact the genotypes of virus found in the female genital compartment.

To develop effective intervention strategies that prevent breast milk transmission of human immunodeficiency virus (HIV), we must understand the specific viral properties and mechanisms responsible for infant infection. We have used lactating rhesus macaques infected with a pathogenic simian immunodeficiency virus (SIV) stock to analyze the viral genotypes expressed in plasma and milk throughout the disease course and to identify those variants ultimately transmitted to infants through breastfeeding. In these studies we observed mother-to-infant transmission of SIV/Delta(B670) by eight females during the chronic phase of disease, and we analyzed by heteroduplex tracking assays and sequence analysis the distribution and fluctuations in viral genotypes expressed. Each female expressed multiple V1 envelope genotypes in milk near the time of transmission, while a single genotype was found in each of the infants. Variants transmitted to infants were not expressed throughout the maternal disease course but were only detected near the time of transmission. The emergence of the transmitted genotype in the dam typically occurred in plasma before milk and was coincident with increased milk viral loads. Transmitted genotypes tended to be longer and more glycosylated and had a less negative charge over the V1 region compared to viral genotypes expressed in milk but not transmitted. These observations demonstrate that specific viral genotypes are selectively transmitted to infants through breastfeeding and support the hypothesis that transmission occurs as genotypes adapt for efficient expression in milk.

As a model of breast milk transmission of HIV, we characterized humoral immune responses in the milk and plasma of 14 female rhesus macaques with suckling infants. Total immunoglobulin levels in plasma and milk were similar in all females and could not be correlated with transmission to the infant. These females, however, had elevated milk IgG levels and decreased milk IgA levels as compared with levels in seronegative controls. SIV envelope-specific antibody responses developed similarly in all females over the first 14-28 days after inoculation; however, 2 females had significantly lower titers by 98 days after inoculation. These females, characterized as rapid disease progressors, were the only animals to transmit SIV through breast-feeding during the period of acute viremia (14-21 days after inoculation). The remaining 12 females developed similar levels of high-avidity SIV envelope-specific IgG in plasma and low, but detectable, levels of IgA in milk. Despite similar quantities of antibody in milk, transmission of SIV through breast-feeding occurred in 8 of 12 mother-baby pairs during the chronic phase of disease. These observations are comparable with those for HIV-infected women and indicate that the SIV-macaque model provides a unique resource for deciphering the functional role of antibodies in breast milk transmission of HIV.

Background: The in utero transmission rate of HIV-1 is estimated to be 10-15% in the absence of interventions and breastfeeding. Natural protective mechanisms involving lentivirus-specific antibodies may therefore exist to limit in utero transmission of lentiviruses., Objectives: HIV-1- and SIV-specific immunoglobulin G (IgG) levels in amniotic fluid samples from humans and rhesus macaques were assessed., Study Design: HIV-1- and SIV-specific immunoglobulin G levels, relative to total IgG concentrations in amniotic fluid samples from humans and rhesus macaques, were determined using a quantitative Western blotting procedure. Amniotic fluid from rhesus macaques was tested for the ability to neutralize SIV infection of CEMX174 cells., Results: The levels of HIV-1- and SIV-specific immunoglobulin G, relative to total IgG concentrations in amniotic fluid samples from humans and rhesus macaques, were approximately 3-10-fold higher than in plasma. The ability of antibodies in human amniotic fluid samples to neutralize viral infectivity could not be assessed, because zidovidine was present in the samples. Most amniotic fluid samples from rhesus macques not treated with antiretrovirals were able to neutralize SIV infectivity, except for a sample from a SIV positive rhesus whose infant was infected in utero., Conclusions: Active immunity to HIV-1 resulting in virus-specific antibodies in amniotic fluid exists, and may be a natural barrier to in utero infection. This may provide hope for stimulating neutralizing antibody via vaccine design.

Background: The viral and host factors involved in transmission of HIV through breastfeeding are largely unknown, and intervention strategies are urgently needed to protect at-risk populations. To evaluate the viral and immunological factors directly related to milk transmission of virus, we have evaluated the disease course of Simian Immunodeficiency Virus (SIV) in lactating rhesus macaques (Macaca mulatta) as a model of natural breast milk transmission of HIV., Results: Fourteen lactating macaques were infected intravenously with SIV/DeltaB670, a pathogenic isolate of SIV and were pair-housed with their suckling infants throughout the disease course. Transmission was observed in 10 mother-infant pairs over a one-year period. Two mothers transmitted virus during the period of initial viremia 14-21 days post inoculation (p.i.) and were classified as early transmitters. Peak viral loads in milk and plasma of early transmitters were similar to other animals, however the early transmitters subsequently displayed a rapid progressor phenotype and failed to control virus expression as well as other animals at 56 days p.i. Eight mothers were classified as late transmitters, with infant infection detected at time points in the chronic stage of the maternal SIV disease course (81 to 360 days). Plasma viral loads, CD4+ T cell counts and SIV-specific antibody titers were similar in late transmitters and non-transmitters. Late breast milk transmission, however, was correlated with higher average milk viral loads and more persistent viral expression in milk 12 to 46 weeks p.i. as compared to non-transmitters. Four mothers failed to transmit virus, despite disease progression and continuous lactation., Conclusion: These studies validate the SIV-infected rhesus macaque as a model for breast milk transmission of HIV. As observed in studies of HIV-infected women, transmission occurred at time points throughout the period of lactation. Transmission during the chronic stage of SIV-infection correlated with a threshold level of virus expression as well as more persistent shedding in milk. This model will be a valuable resource for deciphering viral and host factors responsible for transmission of HIV through breastfeeding.

To decipher the mechanisms involved in oral transmission of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) through breast-feeding, we have developed an animal model using SIV-infected lactating rhesus macaques (Macaca mulatta) and their infants. Five of eight macaque infants became infected during a 10-month study course after SIV inoculation of lactating dams. In a second study, three of four chronically infected female macaques transmitted virus to their infants through breast-feeding within 4 months of birth. Transmission of virus to infants did not correlate with viral loads in either milk or plasma. Infants were infected with homogeneous virus populations, while milk samples near the time of transmission were more diverse. These studies suggest that specific viral phenotypes are selectively transmitted through breast-feeding.

Five rhesus monoclonal antibodies (RhMAbs) were produced by rhesus EBV transformation of peripheral blood B cells from a rhesus macaque that had been asymptomatically infected with an attenuated, macrophage-tropic SIV strain, 17E-Cl. These MAbs recognized conformation-dependent epitopes on SIV gp120 and could not be mapped using synthetic peptides. All five RhMAbs were able to neutralize the vaccine strain and a heterologous isolate, SIV/DeltaB670. The RhMAbs did not cross-react with HIV-2; by contrast, four human MAbs derived from an HIV-2-infected person were broadly cross-reactive with both SIV and HIV-2 gp120s. Cross-competition analysis indicated that the five RhMAbs could be placed in two groups recognizing two nonoverlapping epitopes; while the HMAbs were placed in two additional competition groups. Binding of the three group I RhMAbs (1.7F, 3.11B, and 1.10A) as well as HMAb 17A was shown to be sensitive to specific amino acid alterations in V4 occurring in natural env variants. The results of this study demonstrate that RhEBV transformation provides a means to probe rhesus antibody responses to SIV infection at the monoclonal level. RhMAbs will allow structural and functional studies of envelope glycoprotein determinants that elicit protective immune responses against SIV.

Genetically distinct lentiviruses constitute a quasispecies population that can evolve in response to selective forces. To move beyond characterization of the population as a whole to the behavior of individual members, we devised an in situ hybridization approach that uses genotype-specific probes. We used probes that detect simian immunodeficiency viruses (SIV) that differ in sequence in the V1 region of the surface envelope glycoprotein (env) gene to investigate the replication and cellular tropisms of four viral variants in the tissues of infected rhesus macaques. We found that the V1 genotypic variants replicated in spatially defined patterns and to different extents at each anatomic site. The two variants that replicated most extensively in animals with AIDS were detected in both macrophages and T lymphocytes in tissues. By extension of this approach, it will be possible to investigate the role of individual lentiviruses in a quasispecies in pathogenesis and to evaluate the effects of antiviral or immunotherapeutic treatment on select members of a quasispecies.

To investigate the viral and host factors that contribute to neurological disease, nine macaques were intravenously co-inoculated with SIV/DeltaB670, a primary isolate of SIV consisting of at least 21 different genotypes, and SIV/17E-Fr, a neurovirulent recombinant clone. CD4+ cell counts and antigenemia were measured throughout infection. The SIV env V1 region was amplified from brain and peripheral blood mononuclear cell DNA to compare the genotypes present in brain and blood. Seven of the 9 macaques (78%) developed typical SIV-associated neurological lesions classified as severe (4 macaques), moderate (2 macaques), or mild (1 macaque) with a mean time to euthanasia of 7 months. Macaques with severe neurological lesions progressed more rapidly, with a mean time to euthanasia of 3-6 months. SIV/17E-Fr was detected in brain homogenates from all four macaques with severe encephalitis, and in three of the four, SIV/17E-Fr was the only genotype identified in the central nervous system. Macaques with less severe or no neurological lesions usually had one of various genotypes of SIV/DeltaB670 in brain. A variety of genotypes of SIV/DeltaB670 and SIV/17E-Fr were detected in peripheral blood mononuclear cells throughout infection. Macaques with severe neurological lesions had the most precipitous declines in CD4+ cell counts, the highest levels of antigenemia, and the greatest expression of viral RNA and protein in the central nervous system. Macaca nemestrina were more likely to develop severe neurological lesions than M. mulatta or M. fascicularis (P = 0.048). This study demonstrated that neurovirulent strains within the virus swarm can selectively enter and become established in the central nervous system and that the neurological lesions that develop are correlated with the development of host immunosuppression. The species differences in severity of neurological lesions seen in this study suggest that host factors are also important in determining the outcome of lentiviral infection.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.