A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

The Impact of SIV-Induced Immunodeficiency on Clinical Manifestation, Immune Response, and Viral Dynamics in SARS-CoV-2 Coinfection.

Persistent and uncontrolled SARS-CoV-2 replication in immunocompromised individuals has been observed and may be a contributing source of novel viral variants that continue to drive the pandemic. Importantly, the effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. Here we conducted a pilot study wherein two pigtail macaques (PTM) chronically infected with SIVmac239 were exposed to SARS-CoV-2 and monitored for six weeks for clinical disease, viral replication, and viral evolution, and compared to our previously published cohort of SIV-naïve PTM infected with SARS-CoV-2. At the time of SARS-CoV-2 infection, one PTM had minimal to no detectable CD4+ T cells in gut, blood, or bronchoalveolar lavage (BAL), while the other PTM harbored a small population of CD4+ T cells in all compartments. Clinical signs were not observed in either PTM; however, the more immunocompromised PTM exhibited a progressive increase in pulmonary infiltrating monocytes throughout SARS-CoV-2 infection. Single-cell RNA sequencing (scRNAseq) of the infiltrating monocytes revealed a less activated/inert phenotype. Neither SIV-infected PTM mounted detectable anti-SARS-CoV-2 T cell responses in blood or BAL, nor anti-SARS-CoV-2 neutralizing antibodies. Interestingly, despite the diminished cellular and humoral immune responses, SARS-CoV-2 viral kinetics and evolution were indistinguishable from SIV-naïve PTM in all sampled mucosal sites (nasal, oral, and rectal), with clearance of virus by 3-4 weeks post infection. SIV-induced immunodeficiency significantly impacted immune responses to SARS-CoV-2 but did not alter disease progression, viral kinetics or evolution in the PTM model. SIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants.

Comparison of fine-needle aspiration techniques.

BACKGROUND: Fine-needle aspiration (FNA) has been reported since 1912 beginning with the use of trocars and other specialized instruments that were impractical. Since then, FNA has proven to be a successful alternative technique to excisional biopsy for some assays despite a few limitations. METHODS: In this study, we compared four different techniques for FNA in rhesus macaques by evaluating total live cells recovered and cell viability using a standard 6 mL syringe and 1.5-inch 22-gauge needle. RESULTS: Technique B which was the only technique in which the needle was removed from the syringe after collection of the sample to allow forced air through the needle to expel the contents into media followed by flushing of the syringe and needle resulted in the highest total cell count and second highest cell viability in recovered cells. CONCLUSION: Based on our results, Technique B appears to be the superior method.

Duration of antiretroviral therapy impacts the degree of residual SIV infection in the gut in long-term non-progressing Chinese rhesus macaques.

The gut is a major reservoir in HIV-infected individuals on antiretroviral therapy (ART) and in long-term non-progressors (LTNPs). Whether ART reduces gut infection and reservoirs in LTNPs is unknown. Herein, SIV-infected LTNP Rhesus macaques were treated with short- or long-term ART, and SIV envelope gp120 sequences obtained from single genome amplification were analyzed before and after ART in peripheral blood and the intestine. Although ART does not eliminate SIV in these LTNPs, a longer ART period dramatically reduces SIV infection in the gut. This study highlights the importance of long-term ART in LTNPs to minimize gut infection and prolong remission.

Long-primed germinal centres with enduring affinity maturation and clonal migration.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

Neuroinflammatory Profiling in SIV-Infected Chinese-Origin Rhesus Macaques on Antiretroviral Therapy.

The central nervous system (CNS) HIV reservoir is an obstacle to achieving an HIV cure. The basal ganglia harbor a higher frequency of SIV than other brain regions in the SIV-infected rhesus macaques of Chinese-origin (chRMs) even on suppressive combination antiretroviral therapy (ART). Since residual HIV/SIV reservoir is associated with inflammation, we characterized the neuroinflammation by gene expression and systemic levels of inflammatory molecules in healthy controls and SIV-infected chRMs with or without ART. CCL2, IL-6, and IFN-γ were significantly reduced in the cerebrospinal fluid (CSF) of animals receiving ART. Moreover, there was a correlation between levels of CCL2 in plasma and CSF, suggesting the potential use of plasma CCL2 as a neuroinflammation biomarker. With higher SIV frequency, the basal ganglia of untreated SIV-infected chRMs showed an upregulation of secreted phosphoprotein 1 (SPP1), which could be an indicator of ongoing neuroinflammation. While ART greatly reduced neuroinflammation in general, proinflammatory genes, such as IL-9, were still significantly upregulated. These results expand our understanding of neuroinflammation and signaling in SIV-infected chRMs on ART, an excellent model to study HIV/SIV persistence in the CNS.

Chronic binge alcohol and ovariectomy-mediated impaired insulin responsiveness in SIV-infected female 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-ß 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 SIVmac251, 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.

Co-immunization of DNA and Protein in the Same Anatomical Sites Induces Superior Protective Immune Responses against SHIV Challenge.

We compare immunogenicity and protective efficacy of an HIV vaccine comprised of env and gag DNA and Env (Envelope) proteins by co-administration of the vaccine components in the same muscles or by separate administration of DNA + protein in contralateral sites in female rhesus macaques. The 6-valent vaccine includes gp145 Env DNAs, representing six sequentially isolated Envs from the HIV-infected individual CH505, and matching GLA-SE-adjuvanted gp120 Env proteins. Interestingly, only macaques in the co-administration vaccine group are protected against SHIV CH505 acquisition after repeated low-dose intravaginal challenge and show 67% risk reduction per exposure. Macaques in the co-administration group develop higher Env-specific humoral and cellular immune responses. Non-neutralizing Env antibodies, ADCC, and antibodies binding to FcγRIIIa are associated with decreased transmission risk. These data suggest that simultaneous recognition, processing, and presentation of DNA + Env protein in the same draining lymph nodes play a critical role in the development of protective immunity.

Beneficial Effects of Human Anti-Interleukin-15 Antibody in Gluten-Sensitive Rhesus Macaques with Celiac Disease.

Overexpression of interleukin-15 (IL-15) is linked with immunopathology of several autoimmune disorders including celiac disease. Here, we utilized an anti-human IL-15 antibody 04H04 (anti-IL-15) to reverse immunopathogenesis of celiac disease. Anti-IL-15 was administered to six gluten-sensitive rhesus macaques with celiac disease characteristics including gluten-sensitive enteropathy (GSE), and the following celiac-related metrics were evaluated: morphology (villous height/crypt depth ratio) of small intestine, counts of intestinal intraepithelial lymphocytes, IFN-γ-producing CD8+ and CD4+ T cells, plasma levels of anti-gliadin and anti-intestinal tissue transglutaminase IgG antibodies, as well as peripheral effector memory (CD3+CD28-CD95+) T cells. Anti-IL-15 treatment reversed the clinically relevant disease endpoints, intraepithelial lymphocyte counts, and villous height/crypt depth ratios within jejunal biopsies to normal levels (P < 0.001). Additionally, intestinal CD8+ and CD4+ T cell IFN-γ production was reduced (P < 0.05). Extra-intestinally, anti-IL-15 treatment reduced peripheral NK cell counts (P < 0.001), but otherwise, non-NK peripheral lymphocytes including effector memory T cells and serum blood chemistry were unaffected. Overall, providing the beneficial disease-modulatory and immunomodulatory effects observed, anti-IL-15 treatment might be considered as a novel therapy to normalize intestinal lymphocyte function in celiac disease patients with GSE.

Hydrocephalus after Intrathecal Administration of Dextran to Rhesus Macaques (Macaca mulatta).

Dextrans have been used extensively as medical therapies and labeling agents in biomedical research to investigate the blood-brain barrier and CSF flow and absorption. Adverse effects from dextrans include anaphylactic reaction and dilation of the cerebral ventricles due to administration into the subarachnoid space. This retrospective study describes 51 rhesus macaques (Macaca mulatta) that received dextran intrathecally. The purpose of intrathecal administration was to enable detection of long-lived, dextran-labeled macrophages and to study monocyte-macrophage turnover in the CNS of SIV- or SHIV- infected and uninfected animals by using immunofluorescence. Of the 51 dextran-treated macaques, 8 that received dextran diluted in saline developed hydrocephalus; 6 of these 8 animals exhibited neurologic signs. In contrast, none of the macaques that received intrathecal dextran diluted in PBS developed hydrocephalus. These data suggest the use of saline diluent and the duration of dextran exposure as potential factors contributing to hydrocephalus after intrathecal dextran in rhesus macaques.

Pseudoaneurysm and Arteriovenous Fistula in a Rhesus Macaque (Macaca mulatta).

An 8-y-old female rhesus macaque (Macaca mulatta) presented for swelling of the left lower limb distal to the inguinal region and associated with the femoral artery. Physical and ultrasound examinations suggested an arteriovenous fistula combined with a pseudoaneurysm. After review of possible treatment options, we determined that open surgical repair was the best course of action. The pseudoaneurysm and arteriovenous fistula were surgically resected, and the macaque recovered without complication.

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.

Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Ethanol impairs mucosal immunity against Streptococcus pneumoniae infection by disrupting interleukin 17 gene expression.

Acute ethanol intoxication suppresses the host immune responses against Streptococcus pneumoniae. As interleukin 17 (IL-17) is a critical cytokine in host defense against extracellular pathogens, including S. pneumoniae, we hypothesized that ethanol impairs mucosal immunity against this pathogen by disrupting IL-17 production or IL-17 receptor (IL-17R) signaling. A chronic ethanol feeding model in simian immunodeficiency virus (SIV)-infected rhesus macaques and acute ethanol intoxication in a murine model were used. Transcriptome analysis of bronchial brushes in the nonhuman primate model showed downregulation of the expression of IL-17-regulated chemokines in ethanol-fed animals, a finding also replicated in the murine model. Surprisingly, recombinant CXCL1 and CXCL5 but not IL-17 or IL-23 plus IL-1ß rescued bacterial burden in the ethanol group to control levels. Taken together, the results of this study suggest that ethanol impairs IL-17-mediated chemokine production in the lung. Thus, exogenous luminal restoration of IL-17-related chemokines, CXCL1 and CXCL5, improves host defenses against S. pneumoniae.

Transmitted/founder simian immunodeficiency virus envelope sequences in vesicular stomatitis and Semliki forest virus vector immunized rhesus macaques.

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID50. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.

Effects of alcohol consumption on antigen-specific cellular and humoral immune responses to SIV in rhesus macaques.

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.

Effect of bacterial pneumonia on lung simian immunodeficiency virus (SIV) replication in alcohol consuming SIV-infected rhesus macaques.

BACKGROUND: Opportunistic infections in human immunodeficiency virus (HIV)-infected persons have been shown to increase the rate of HIV replication. In populations where prophylaxis against Pneumocystis pneumonia is utilized, bacterial pneumonia is now the leading cause of lower respiratory tract infection in HIV+ patients. Our prior studies have shown that chronic alcohol consumption in demarcated simian immunodeficiency virus (SIV)-infected rhesus macaques increases plasma viral load set point and accelerates progression to end-stage acquired immune deficiency syndrome. While chronic alcohol abuse is well known to increase the incidence and severity of bacterial pneumonia, the impact of alcohol consumption on local and systemic SIV/HIV burden during lung infection is unknown. Therefore, we utilized the macaque SIV infection model to examine the effect of chronic ethanol (EtOH) feeding on SIV burden during the course of pulmonary infection with Streptococcus pneumoniae, the most commonly identified etiology of bacterial pneumonia in HIV+ and HIV- persons in developed countries. METHODS: Alcohol was administered starting 3 months before SIVmac251 inoculation to the end of the study via an indwelling intragastric catheter to achieve a plasma alcohol concentration of 50 to 60 mM. Control animals received isocaloric sucrose. Four months after SIV infection, the right lung was inoculated with 2 × 10(6) CFU S. pneumoniae. RESULTS: Leukocyte recruitment into the lung, pulmonary bacterial clearance, and clinical course were similar between EtOH and control groups. While plasma SIV viral load was similar between groups postpneumonia, chronic EtOH-fed macaques showed a prolonged increase in SIV RNA in bronchoalveolar lavage fluid. Alveolar macrophages isolated from EtOH-fed macaques 1 day post-pneumonia showed greater nuclear factor kappa beta (NF-κB) activation. CONCLUSIONS: This study indicates that chronic EtOH feeding results in enhanced local, but not systemic, SIV replication following pneumococcal pneumonia. Increased NF-κB activity in the setting of chronic EtOH ingestion may play a mechanistic role in this observation.

Excision of femoral head and neck for treatment of coxofemoral degenerative joint disease in a rhesus macaque (Macaca mulatta).

Nonhuman primates are a valuable model for osteoarthritis. Osteoarthritis has been extensively studied in nonhuman primates in both naturally occurring and induced disease states. However, little published information describes naturally occurring osteoarthritis of the coxofemoral joints of nonhuman primates. We report a case of naturally occurring coxofemoral joint osteoarthritis in a rhesus macaque. This case radiographically resembled hip dysplasia reported in other species and demonstrated a rapid progression in severity of lameness, with accompanying loss of muscle mass in the affected limb. We excised the femoral head and neck to alleviate the pain that accompanied the osteoarthritis. Physical therapy was initiated, and dual-energy X-ray absorptiometry and video recordings were performed to evaluate the macaque's response to surgical intervention. By 3 mo postoperatively, the macaque had regained full use of the affected limb.

Disrupted anabolic and catabolic processes may contribute to alcohol-accentuated SAIDS-associated wasting.

BACKGROUND: Alcohol abuse is a comorbid factor in many human immunodeficiency virus (HIV)-infected patients. Previously, we demonstrated that chronic binge alcohol accentuates loss of body mass at terminal stage of simian immunodeficiency virus (SIV) infection. The purpose of this study was to investigate changes in pathways that may contribute to muscle wasting in chronic binge alcohol-fed SIV-infected macaques. METHODS: The impact of chronic binge alcohol during SIV infection on insulin signaling and the ubiquitin (Ub)-proteasome system-regulators of protein synthesis and degradation-was examined in SIV-infected macaques. RESULTS: SIV infection induced an inflammatory and pro-oxidative milieu in skeletal muscle, which was associated with decreased insulin-stimulated phosphatidylinositol 3-kinase (PI-3k) activity and upregulated gene expression of mTOR and atrogin-1, and protein expression of Ub-proteasome system 19S base. Chronic binge alcohol accentuated the skeletal muscle pro-oxidative milieu and 19S base expression. Additionally, chronic binge alcohol increased skeletal muscle protein expression of protein-tyrosine phosphatase 1B (a negative regulator of insulin signaling) and 19S proteasome regulator non-ATPase (Rpn) 6 subunit and Rpn12, and suppressed PI-3K activity. Animals that were alcohol-fed and SIV-infected for >15 months had increased Ub-proteasome system activity. CONCLUSIONS: These data suggest negative modulation of insulin signaling coupled with enhanced Ub-proteasome system activity may be central mechanisms underlying chronic binge alcohol-induced accentuation of SIV-associated muscle wasting.