Abstract Tuberculosis (TB) remains a leading cause of death from infectious disease worldwide, underscoring the urgent need for vaccines with greater and more consistent efficacy than Bacille Calmette–Guérin (BCG). We previously showed that mucosal vaccination with an isogenic Mycobacterium tuberculosis ( Mtb ) mutant lacking the stress-response transcription factor SigH ( ΔsigH ) prevents pulmonary TB in two macaque species. In the absence of SigH, Mtb is unable to effectively counter host oxidative stress. Vaccinated macaques were notably protected from disease, exhibiting an absence of granulomatous pathology together with the formation of lymphoid follicles and robust antigen-specific CD4 + and CD8 + T cell responses, identifying ΔsigH as a promising live-attenuated TB vaccine candidate. The ΔsigH mutant used in prior studies was generated in the Mtb CDC1551 background, a commonly used laboratory strain for challenge studies. Here, we evaluated whether ΔsigH -mediated protection extends to heterologous challenge with the more virulent Mtb Erdman strain. Mucosal ΔsigH vaccination conferred significant protection against heterologous challenge, markedly reducing pulmonary bacterial burden and TB-associated pathology. Longitudinal high-resolution PET/CT imaging demonstrated that aerosol ΔsigH vaccination induced robust inducible bronchus-associated lymphoid tissue (iBALT) responses in the lung. Unlike granulomas, these iBALT structures resolved over time while remaining associated with protection against subsequent Mtb challenge. Protection of highly susceptible rhesus macaques against virulent heterologous Mtb challenge following aerosol ΔsigH vaccination supports the further preclinical development of ΔsigH -based live-attenuated TB vaccines and highlights iBALT induction as a potential correlate and mechanistic driver of protective immunity against TB.

Background:Aerosol exposure of non-human primates (NHPs) to Mycobacterium tuberculosis (Mtb) typically results in discrete sites of inflammation of the lung that is detectable by 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18 F-FDG)-based PET/CT scans. Such scans are often analyzed using software such as Invicro VivoQuant or OsiriX as 3D images by manual labeling sites of PET signal using 2D slices and by reporting maximal standardized uptake value (SUV max) either of the whole lung or of individual lesions. Methods:Here we propose a pipeline for analysis of the same PET/CT scans using Imaris, a proprietary software typically used for analysis of fluorescent microscopy data. Results:We show that by using locations of spine vertebra (denoted as "landmarks") we can align serials scans of the same animal, and by using automated (with some manual corrections) image segmentation of PET scans in 3D as "surfaces", we can accurately define location of all sites of inflammation in the lung and lung-associated thoracic lymph nodes (LNs). We show that there is an excellent correlation between individual lesion's SUVmax determined by VivoQuant and maximum intensity determined by Imaris suggesting utility of this approach. Imaris also provides wealth of additional information for each of the identified lesions such as volume, location, shape, surface area, and others, and each lesion can be exported in Virtual Reality file format (.wrl). Conclusion:Our novel methodology allows for detailed and rigorous analyses of how features of PET-defined lesions in Mtb-exposed monkeys evolve over time and correlate with the outcome of infection and/or treatment.

Central nervous system (CNS) macrophages play key roles in viral neuropathogenesis and immune surveillance, yet their trafficking and trafficking dynamics between the CNS and peripheral lymphoid tissues remain poorly understood. To address this gap, we used intracisternal (i.c.) injection of fluorescent-superparamagnetic iron oxide nanoparticles (SPION) in SIV-infected macaques that we found labeled CNS CD68+CD163+CD206+ perivascular, meningeal, and choroid plexus (CP) macrophages. SPION+CD163+ macrophages were also detected in the optic nerves and cribriform area-potential routes of macrophage egress from the CNS. CD163+SPION+ macrophages labeled in the CNS were identified in the deep cervical lymph node (dCLN) and dorsal root ganglia (DRG). SPION+ CD163+ macrophages appeared in both the CNS and the periphery as early as 24 h post i.c. inoculation, decreasing over time in the CNS of non-infected animals but accumulating with SIV infection. A trend toward greater numbers of SPION + macrophages that traffic out of the CNS was observed in non-infected animals, a pattern not seen with SIV infection. Productively infected SPION+ cells were found in the CNS and dCLN of infected animals 7-28 days post i.c. injection. These findings support a model in which SPION + macrophages, some of which harbor virus, traffic between the CNS and peripheral lymphoid tissues. This is discussed with regard to HIV infection of the CNS and reseeding of the periphery with CNS virus.

To radically diminish tuberculosis (TB) incidence and mortality by 2035, as set out by the WHO End TB Strategy, there is a desperate need for improved TB therapies and a more effective vaccine against the deadly pathogen Mycobacterium tuberculosis. Aerosol vaccination with the MtbΔsigH mutant protects 2 species of nonhuman primates against lethal TB challenge by invoking vastly superior T and B cell responses in the lungs through superior antigen presentation and interferon conditioning. Since the Geneva Consensus on essential steps toward the development of live mycobacterial vaccines recommends that live TB vaccines incorporate at least 2 independent gene knockouts, we have now generated several rationally designed, double-knockout (DKO) and triple-knockout (TKO) mutants in Mtb, each containing the ΔsigH deletion. Here, we report preclinical studies in the rhesus macaque model of aerosol infection and SIV/HIV coinfection, aimed at assessing the safety of these MtbΔsigH-based DKOs and TKOs. We found that most of these mutant strains were attenuated in both immunocompetent and SIV-coinfected macaques, and combinatorial infection with these generated strong cellular immune responses in the lung, akin to MtbΔsigH. Aerosol infection with these KO strains elicited inducible bronchus-associated lymphoid tissue, which is a correlate of protection from TB.

The effects of antiretroviral therapy (ART) and treatment interruption on myeloid cell egress from the central nervous system (CNS) during human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection remain poorly defined. We hypothesized that CNS macrophages normally traffic out of the CNS, accumulate during viral infection and inflammation, and are released as inflammation resolves. To test this, we administered intracisternal (i.c.) injections of two different colored fluorescent superparamagnetic iron oxide nanoparticles (SPION) to SIV-infected macaques either early in infection (12-14 dpi) or 30 days before necropsy. SPION are preferentially taken up by perivascular, meningeal, and choroid plexus macrophages, enabling us to track macrophage turnover, infection, and migration. In non-infected macaques, SPION+ macrophages trafficked from the CNS to peripheral sites including the deep cervical lymph node (dCLN), lumbar lymph nodes, spleen, and dorsal root ganglia (DRG). With SIV infection, these cells accumulated in the CNS and showed reduced peripheral trafficking. ART decreased the number of SPION+ perivascular macrophages, and to a lesser extent, meningeal or choroid plexus macrophages. After ART interruption, SPION+ perivascular and choroid plexus macrophage numbers remained stable, whereas SPION+ meningeal macrophages increased. ART eliminated SIV-RNA+ perivascular macrophages, leaving few- scattered SIV-RNA+ cells in the meninges and choroid plexus. Following ART interruption, perivascular macrophages remained virus-negative, but scattered viral RNA+ meningeal macrophages persisted. In non-infected macaques, SPION+ macrophages trafficked to the dCLN, spleen, and DRG, but this trafficking diminished with SIV infection and AIDS with SIVE. Importantly, SIV-RNA+ SPION+ macrophages that exited the CNS were cleared by ART and did not reappear after treatment interruption. Using two differently colored SPION to assess establishment of CNS viral reservoirs, we observed higher numbers of early-labeled macrophages within and outside the CNS in animals with AIDS and SIVE, ART treatment, and ART interruption. These findings support a model in which SIV-infected perivascular macrophages seed an early CNS viral reservoir, while the meninges and choroid plexus undergo continual viral seeding during infection. ART reduces trafficking of infected macrophages out of the CNS and clears the perivascular macrophage reservoir, but SIV-RNA+ meningeal macrophages can persist, in low numbers, and can rebound after ART interruption.

We use intracisternal (i.c.) injection of fluorescent-superparamagnetic iron oxide nanoparticles (SPION) in SIV-infected monkeys that labeled CNS CD68-CD163-CD206 perivascular, meningeal, and choroid plexus (CP) macrophages. SPION + CD163 + macrophages are also found in the optic nerves, nasal septum, and cribriform plate - potential sites-paths of macrophage exit. Outside the CNS CD163 + SPION + macrophages labeled in the CNS are in deep cervical lymph node (dCLN), spleen, dorsal root ganglia (DRG). CD163 + SPION + CNS macrophages are abundant 24 hours p.i. in normal animals and decrease over time without CNS inflammation, but accumulate with SIV infection and inflammation. Greater numbers of SPION + macrophages traffic out of the CNS in normal animals and decrease with infection. Productively infected, SPION + macrophages are found in the CNS and dCLN, spleen, and DRG of infected animals 7-14 days post i.c. injection. These data are consistent with SPION + macrophages, some of which are viral infected, trafficking out of the CNS.

Most HIV-negative individuals exposed to Mycobacterium tuberculosis (Mtb) control infection as latent TB infection (LTBI), but HIV co-infection greatly increases progression to tuberculosis (TB), the leading cause of death in people living with HIV (PLHIV). Although combination antiretroviral therapy (cART) reduces LTBI reactivation, immune control of Mtb is not fully restored, as shown by persistent TB incidence in PLHIV on cART. In macaques, skewed pulmonary effector memory CD4⁺ T-cell (TEM) responses and new TB lesions persist despite cART. We hypothesize that concurrent anti-TB therapy with cART would improve bacterial control and immune restoration compared to cART alone. Using rhesus macaques (RM) with LTBI and Simian Immunodeficiency Virus (SIV) co-infection, we tested three months of weekly isoniazid and rifapentine (3HP) plus daily cART. Concurrent cART+3HP improves clinical and microbiological outcomes but fails to fully restore lung CD4⁺ T-cell immunity. Treated RMs retain caseous granulomas with high FDG uptake and incomplete CD4⁺ T-cell reconstitution, marked by persistent activation, exhaustion, and inflammation. CD4⁺ TEM cells remain depleted. Concurrent therapy induces Type I IFN signatures and enhances Mtb-specific TH1/TH17—but reduces TNFα—responses. These findings reveal persistent pulmonary immune defects underlying TB risk in HIV co-infection and identify potential targets for host-directed adjunctive therapies. The basis for inefficient Mtb control in PLWH despite treatment is unclear. Here, the authors show that combined TB/HIV therapy in Mtb/SIV co-infected macaques reduces bacterial and viral burden and improved lung pathology, but failed to fully restore immune balance, revealing persistent inflammation.

Tuberculosis (TB) remains the leading cause of death due to infection globally. Bacillus Calmette Guérin (BCG), a live attenuated bacterial strain, is the only available TB vaccine, but it has poor efficacy in preventing pulmonary TB in adults. There are advantages associated with the BCG platform however, including a remarkable safely profile, billions of administered doses and a public health ecosystem associated with its production, administration and care. A recombinant/modified BCG (rBCG/modBCG) anti-TB vaccine candidate would be able to leverage these advantages while improving on efficacy. BCG-STING is a recombinant BCG strain which overexpresses the mycobacterial diadenylate cyclase disA gene leading to the release of ~15-fold greater levels of the endogenous small molecule STING agonist cyclic di-AMP. We evaluated vaccination with BCG-STING compared to its parental BCG-WT strain in rhesus macaques challenged with virulent Mycobacterium tuberculosis (Mtb). BCG-STING given intradermally was well-tolerated, and during life serial BAL samples showed that BCG-STING vaccinated animals had lower Mtb CFU counts than those receiving BCG-WT. At necropsy, BCG-STING vaccinated animals had significantly lower Mtb lung CFU counts, and a higher percentage of sterile lobes and granuloma lesions than the BCG-WT recipients. The enhanced protection observed in BCG-STING vaccinated animals was associated with significant elevations of antigen-specific CD4+ and CD8+ T cells. Modifying BCG to overexpress a small molecule recognized by the innate immune system significantly improves protection and cell-mediated immune responses against TB the non-human primate (NHP) model.

Chronic immune activation promotes tuberculosis (TB) reactivation in the macaque Mycobacterium tuberculosis (M. tuberculosis)/SIV coinfection model. Initiating combinatorial antiretroviral therapy (cART) early lowers the risk of TB reactivation, but immune activation persists. Studies of host-directed therapeutics (HDTs) that mitigate immune activation are, therefore, required. Indoleamine 2,3, dioxygenase (IDO), a potent immunosuppressor, is one of the most abundantly induced proteins in NHP and human TB granulomas. Inhibition of IDO improves immune responses in the lung, leading to better control of TB, including adjunctive to TB chemotherapy. The IDO inhibitor D-1 methyl tryptophan (D1MT) is, therefore, a bona fide TB HDT candidate. Since HDTs against TB are likely to be deployed in an HIV coinfection setting, we studied the effect of IDO inhibition in M. tuberculosis/SIV coinfection, adjunctive to cART. D1MT is safe in this setting, does not interfere with viral suppression, and improves the quality of CD4+ and CD8+ T cell responses, including reconstitution, activation and M. tuberculosis–specific cytokine production, and access of CD8+ T cells to the lung granulomas; it reduces granuloma size and necrosis, type I IFN expression, and the recruitment of inflammatory IDO+ interstitial macrophages (IMs). Thus, trials evaluating the potential of IDO inhibition as HDT in the setting of cART in M. tuberculosis/HIV coinfected individuals are warranted.

The majority of Human Immunodeficiency Virus (HIV) negative individuals exposed to Mycobacterium tuberculosis (Mtb) control the bacillary infection as latent TB infection (LTBI). Co-infection with HIV, however, drastically increases the risk to progression to tuberculosis (TB) disease. TB is therefore the leading cause of death in people living with HIV (PLWH) globally. Combinatorial antiretroviral therapy (cART) is the cornerstone of HIV care in humans and reduces the risk of reactivation of LTBI. However, the immune control of Mtb infection is not fully restored by cART as indicated by higher incidence of TB in PLWH despite cART. In the macaque model of co-infection, skewed pulmonary CD4+ TEM responses persist, and new TB lesions form despite cART treatment. We hypothesized that regimens that concurrently administer anti-TB therapy and cART would significantly reduce TB in co-infected macaques than cART alone, resulting in superior bacterial control, mitigation of persistent inflammation and lasting protective immunity. We studied components of TB immunity that remain impaired after cART in the lung compartment, versus those that are restored by concurrent 3 months of once weekly isoniazid and rifapentine (3HP) and cART in the rhesus macaque (RM) model of LTBI and Simian Immunodeficiency Virus (SIV) co-infection. Concurrent administration of cART + 3HP did improve clinical and microbiological attributes of Mtb/SIV co-infection compared to cART-naïve or -untreated RMs. While RMs in the cART + 3HP group exhibited significantly lower granuloma volumes after treatment, they, however, continued to harbor caseous granulomas with increased FDG uptake. cART only partially restores the constitution of CD4 + T cells to the lung compartment in co-infected macaques. Concurrent therapy did not further enhance the frequency of reconstituted CD4+ T cells in BAL and lung of Mtb/SIV co-infected RMs compared to cART, and treated animals continued to display incomplete reconstitution to the lung. Furthermore, the reconstituted CD4+ T cells in BAL and lung of cART + 3HP treated RMs exhibited an increased frequencies of activated, exhausted and inflamed phenotype compared to LTBI RMs. cART + 3HP failed to restore the effector memory CD4+ T cell population that was significantly reduced in pulmonary compartment post SIV co-infection. Concurrent therapy was associated with the induction of Type I IFN transcriptional signatures and led to increased Mtb-specific TH1/TH17 responses correlated with protection, but decreased Mtb-specific TNFa responses, which could have a detrimental impact on long term protection. Our results suggest the mechanisms by which Mtb/HIV co-infected individuals remain at risk for progression due to subsequent infections or reactivation due of persisting defects in pulmonary T cell responses. By identifying lung-specific immune components in this model, it is possible to pinpoint the pathways that can be targeted for host-directed adjunctive therapies for TB/HIV co-infection.

Intestinal epithelial barrier dysfunction, a hallmark of HIV/SIV infection, persists despite viral suppression by combination antiretroviral therapy (cART). Emerging evidence suggests a critical role for long noncoding RNAs (lncRNAs) in maintaining epithelial homeostasis. We simultaneously profiled lncRNA/mRNA expression exclusively in colonic epithelium (CE) of SIV-infected rhesus macaques (RMs) administered vehicle (VEH) or Δ-9-tetrahydrocannabinol (THC). Relative to controls, fewer lncRNAs were up- or downregulated in CE of THC/SIV compared with VEH/SIV RMs. Importantly, reciprocal expression of the natural antisense lncRNA MMP25-AS1 (up 2.3-fold) and its associated protein-coding gene MMP25 (attracts neutrophils by inactivating alpha-1 anti-trypsin/SERPINA1) (down 2.2-fold) was detected in CE of THC/SIV RMs. Computational analysis verified 2 perfectly matched complementary regions and an energetically stable (normalized binding free energy = –0.2626) MMP25-AS1/MMP25 duplex structure. MMP25-AS1 overexpression blocked IFN-γ–induced MMP25 mRNA and protein expression in vitro. Elevated MMP25 protein expression in CE of VEH/SIV but not THC/SIV RMs was associated with increased infiltration by myeloperoxidase/CD11b++ neutrophils (transendothelial migration) and epithelial CD47 (transepithelial migration) expression. Interestingly, THC administered in combination with cART increased MMP25-AS1 and reduced MMP25 mRNA/protein expression in jejunal epithelium of SIV-infected RMs. Our findings demonstrate that MMP25-AS1 is a potentially unique epigenetic regulator of MMP25 and that low-dose THC can reduce neutrophil infiltration and intestinal epithelial injury potentially by downregulating MMP25 expression through modulation of MMP25-AS1.

The expression of indoleamine 2,3-dioxygenase (IDO), a robust immunosuppressant, is significantly induced in macaque tuberculosis (TB) granulomas, where it is expressed on IFN-responsive macrophages and myeloid-derived suppressor cells. IDO expression is also highly induced in human TB granulomas, and products of its activity are detected in patients with TB. In vivo blockade of IDO activity resulted in the reorganization of the granuloma with substantially greater T cells being recruited to the core of the lesions. This correlated with better immune control of TB and reduced lung M. tuberculosis burdens. To study if the IDO blockade strategy can be translated to a bona fide host-directed therapy in the clinical setting of TB, we studied the effect of IDO inhibitor 1-methyl-d-tryptophan adjunctive to suboptimal anti-TB chemotherapy. While two-thirds of controls and one-third of chemotherapy-treated animals progressed to active TB, inhibition of IDO adjunctive to the same therapy protected macaques from TB, as measured by clinical, radiological, and microbiological attributes. Although chemotherapy improved proliferative T cell responses, adjunctive inhibition of IDO further enhanced the recruitment of effector T cells to the lung. These results strongly suggest the possibility that IDO inhibition can be attempted adjunctive to anti-TB chemotherapy in clinical trials.

The rebound-competent viral reservoir (RCVR), comprised of virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after antiretroviral therapy interruption (ATI), remains the biggest obstacle to the eradication of HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop targeted therapeutic strategies for reducing the RCVR. In this study, barcoded SIVmac239M was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood, lymphoid tissues (LTs, spleen, mesenteric and inguinal lymph nodes), and non-lymphoid tissues (NLTs, colon, ileum, lung, liver, and brain) were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX/RNAscope/ in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy although plasma viral RNA remained < 22 copies/mL. Among the tissues studied, mesenteric and inguinal lymph nodes, and spleen contained viral barcodes detected in plasma, and trended to have higher cell-associated viral loads, higher intact provirus levels, and greater diversity of viral barcodes. CD4+ T cells were the main cell type harboring viral RNA (vRNA) after ATI. Further, T cell zones in LTs showed higher vRNA levels than B cell zones for most animals. These findings are consistent with LTs contributing to virus present in plasma early after ATI.One Sentence Summary:The reemerging of SIV clonotypes at early post-ATI are likely from the secondary lymphoid tissues.

Background Although the advent of combination anti-retroviral therapy (cART) has transformed HIV into a manageable chronic disease, an estimated 30–50% of people living with HIV (PLWH) exhibit cognitive and motor deficits collectively known as HIV-associated neurocognitive disorders (HAND). A key driver of HAND neuropathology is chronic neuroinflammation, where proinflammatory mediators produced by activated microglia and macrophages are thought to inflict neuronal injury and loss. Moreover, the dysregulation of the microbiota–gut–brain axis (MGBA) in PLWH, consequent to gastrointestinal dysfunction and dysbiosis, can lead to neuroinflammation and persistent cognitive impairment, which underscores the need for new interventions. Methods We performed RNA-seq and microRNA profiling in basal ganglia (BG), metabolomics (plasma) and shotgun metagenomic sequencing (colon contents) in uninfected and SIV-infected rhesus macaques (RMs) administered vehicle (VEH/SIV) or delta-9-tetrahydrocannabinol (THC) (THC/SIV). Results Long-term, low-dose THC reduced neuroinflammation and dysbiosis and significantly increased plasma endocannabinoid, endocannabinoid-like, glycerophospholipid and indole-3-propionate levels in chronically SIV-infected RMs. Chronic THC potently blocked the upregulation of genes associated with type-I interferon responses ( NLRC5 , CCL2 , CXCL10 , IRF1 , IRF7 , STAT2 , BST2 ), excitotoxicity ( SLC7A11 ), and enhanced protein expression of WFS1 (endoplasmic reticulum stress) and CRYM (oxidative stress) in BG. Additionally, THC successfully countered miR-142-3p-mediated suppression of WFS1 protein expression via a cannabinoid receptor-1-mediated mechanism in HCN2 neuronal cells. Most importantly, THC significantly increased the relative abundance of Firmicutes and Clostridia including indole-3-propionate ( C. botulinum , C. paraputrificum , and C. cadaveris ) and butyrate ( C. butyricum , Faecalibacterium prausnitzii and Butyricicoccus pullicaecorum ) producers in colonic contents. Conclusion This study demonstrates the potential of long-term, low-dose THC to positively modulate the MGBA by reducing neuroinflammation, enhancing endocannabinoid levels and promoting the growth of gut bacterial species that produce neuroprotective metabolites, like indole-3-propionate. The findings from this study may benefit not only PLWH on cART, but also those with no access to cART and more importantly, those who fail to suppress the virus under cART.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

Background HIV/SIV-associated periodontal disease (gingivitis/periodontitis) (PD) represents a major comorbidity affecting people living with HIV (PLWH) on combination anti-retroviral therapy (cART). PD is characterized by chronic inflammation and dysbiosis. Nevertheless, the molecular mechanisms and use of feasible therapeutic strategies to reduce/reverse inflammation and dysbiosis remain understudied and unaddressed. Methods Employing a systems biology approach, we report molecular, metabolome and microbiome changes underlying PD and its modulation by phytocannabinoids [delta-9-tetrahydrocannabinol (Delta(9)-THC)] in uninfected and SIV-infected rhesus macaques (RMs) untreated (VEH-untreated/SIV) or treated with vehicle (VEH/SIV) or Delta(9)-THC (THC/SIV). Findings VEH- untreated/SIV but not THC/SIV RMs showed significant enrichment of genes linked to anti-viral defense, interferon-beta, NF kappa B, RIG-1, and JAK-STAT signaling. We focused on the anti-microbial DUOX1 and immune activation marker IDO1 that were reciprocally regulated in the gingiva of VEH-untreated/SIV RMs. Both proteins localized to the gingival epithelium and CD163(+) macrophages, and showed differential expression in the gingiva of THC/SIV and VEH/SIV RMs. Additionally, inflammation-associated miR-21, miR-142-3p, miR-223, and miR-125a-5p showed significantly higher expression in the gingiva of VEH/SIV RMs. In human primary gingival epithelial cells, miR-125a-5p post-transcriptionally downregulated DUOX1 and THC inhibited IDO1 protein expression through a cannabinoid receptor-2 mediated mechanism. Interestingly, THC/SIV RMs showed relatively reduced plasma levels of kynurenine, kynurenate, and the neurotoxic quinolinate compared to VEH/SIV RMs at 5 months post SIV infection (MPI). Most importantly, THC blocked HIV/SIV-induced depletion of Firmicutes and Bacteroidetes, and reduced Gammaproteobacteria abundance in saliva. Reduced IDO1 protein expression was associated with significantly (p<0.05) higher abundance of Prevotella, Lactobacillus (L. salivarius, L. buchneri, L. fermentum, L. paracasei, L. rhamnosus, L. johnsonii) and Bifidobacteria and reduced abundance of the pathogenic Porphyromonas cangingivalis and Porphyromonas macacae at 5MPI. Interpretation The data provides deeper insights into the molecular mechanisms underlying HIV/SIV-induced PD and more importantly, the anti-inflammatory and anti-dysbiotic properties of THC in the oral cavity. Overall, these translational findings suggest that phytocannabinoids may help reduce gingival/systemic inflammation, salivary dysbiosis and potentially metabolic disease/syndrome in PLWH on cART and those with no access to cART or do not suppress the virus under cART. Copyright (C) 2021 The Authors. Published by Elsevier B.V.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.

With the advent of the novel SARS-CoV-2, the entire world has been thrown into chaos with severe disruptions from a normal life. While the entire world was going chaotic, the researchers throughout the world were struggling to contribute to the best of their capabilities to advance the understanding of this new pandemic and fast track the development of novel therapeutics and vaccines. While various animal models have helped a lot to understand the basic physiology, nonhman primates have been promising and much more successful in modelling human diseases compared to other available clinical models. Here we describe the different aspects of modelling the SARS-CoV-2 infection in NHPs along with the associated methods used in NHP immunology.