Persistent Viral Reservoirs in Lymphoid Tissues in SIV-Infected Rhesus Macaques of Chinese-Origin on Suppressive Antiretroviral Therapy

Understanding HIV latent reservoirs in tissues is essential for the development of new strategies targeting these sites for eradication. Here, we assessed the size of latent reservoirs and the source of residual viruses in multiple lymphoid tissues of SIV-infected and fully suppressed rhesus macaques of Chinese-origin (cRMs). Eight cRMs were infected with SIVmac251 and treated with tenofovir and emtricitabine daily for 24 weeks initiated 4 weeks post-infection. Four of the eight animals reached sustained full viral suppression with undetectable viremia. The levels of cell-associated SIV DNA varied in peripheral blood mononuclear cells (PBMCs) and multiple lymphoid tissues, but with higher levels in the mesenteric lymph nodes (MesLNs). The levels of cell-associated SIV RNA also varied in different tissues. The higher frequency of viral RNA detection in the MesLNs was also observed by in situ hybridization. Consistently, the infection unit per million cells (IUPM) in the MesLNs was higher than in PBMCs and other tested lymphoid tissues by quantitative viral outgrowth assay (QVOA). Furthermore, env gp120 from tissue SIV RNA was amplified by single genome amplification. Phylogenetic analysis revealed diverse variants from tissues parallel to the viral inoculum in all viral suppressed animals. These results demonstrate that the latency and viral reservoirs in the lymphoid tissues still exist in aviremic macaques under full suppressive therapy. Moreover, the size of viral latent reservoirs differs in various lymphoid tissues with a relatively larger size in the MesLNs

Simian Varicella Virus DNA in Saliva and Buccal Cells After Experimental Acute Infection in Rhesus Macaques

Simian varicella virus (SVV) infection of non-human primates is the counterpart of varicella zoster virus (VZV) infection in humans. To develop non-invasive methods of assessing SVV infection, we tested for the presence of SVV DNA in saliva, as has been documented in human VZV infection, and in buccal cells to determine whether epithelial cells might provide a more reliable source of material for analysis. Five rhesus macaques intratracheally inoculated with SVV all developed varicella with viremia and macular-papular skin rash in 1–2 weeks, which resolved followed by establishment of latency. DNA extracted from longitudinal blood peripheral blood mononuclear cells (PBMCs), saliva and buccal samples collected during acute infection and establishment of latency were analyzed by real-time qPCR. After intratracheal inoculation, viremia was observed, with peak levels of 101–102 copies of SVV DNA in 100 ng of PBMC DNA at 4 and 7 days post inoculation (dpi), which then decreased at 9–56 dpi. In saliva and buccal cells at 7 dpi, 101–104 copies and 101–105 copies of SVV DNA in 100 ng of cellular DNA, respectively, were detected in all the five monkeys. At 9 dpi, saliva samples from only two of the five monkeys contained SVV DNA at 102–103 copies/100 ng of saliva DNA, while buccal cells from all five monkeys showed 100–103 copies of SVV DNA/100 ng of buccal cell DNA. Similar to viremia, SVV DNA in saliva and buccal cells at 11–56 dpi was lower, suggesting clearance of viral shedding. SVV DNA levels were generally higher in buccal cells than in saliva. Our findings indicate that SVV shedding into the oral cavity parallels acute SVV infection and underscore the relevance of both saliva and buccal cell samples to monitor acute varicella virus infection

SARS-CoV-2 infection of the pancreas promotes thrombofibrosis and is associated with new-onset diabetes

Evidence suggests an association between severe acute respiratory syndrome-cornavirus-2 (SARS-CoV-2) infection and the occurrence of new-onset diabetes. We examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), the cell entry factors for SARS-CoV-2, using publicly available single-cell RNA sequencing data sets, and pancreatic tissue from control male and female nonhuman primates (NHPs) and humans. We also examined SARS-CoV-2 immunolocalization in pancreatic cells of SARS-CoV-2-infected NHPs and patients who had died from coronavirus disease 2019 (COVID-19). We report expression of ACE2 in pancreatic islet, ductal, and endothelial cells in NHPs and humans. In pancreata from SARS-CoV-2-infected NHPs and COVID-19 patients, SARS-CoV-2 infected ductal, endothelial, and islet cells. These pancreata also exhibited generalized fibrosis associated with multiple vascular thrombi. Two out of 8 NHPs developed new-onset diabetes following SARS-CoV-2 infection. Two out of 5 COVID-19 patients exhibited new-onset diabetes at admission. These results suggest that SARS-CoV-2 infection of the pancreas may promote acute and especially chronic pancreatic dysfunction that could potentially lead to new-onset diabetes

A cellular trafficking signal in the SIV envelope protein cytoplasmic domain is strongly selected for in pathogenic infection

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

Medical imaging of pulmonary disease in SARS-CoV-2-exposed non-human primates

Chest X-ray (CXR), computed tomography (CT), and positron emission tomography-computed tomography (PET-CT) are noninvasive imaging techniques widely used in human and veterinary pulmonary research and medicine. These techniques have recently been applied in studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-exposed non-human primates (NHPs) to complement virological assessments with meaningful translational readouts of lung disease. Our review of the literature indicates that medical imaging of SARS-CoV-2-exposed NHPs enables high-resolution qualitative and quantitative characterization of disease otherwise clinically invisible and potentially provides user-independent and unbiased evaluation of medical countermeasures (MCMs). However, we also found high variability in image acquisition and analysis protocols among studies. These findings uncover an urgent need to improve standardization and ensure direct comparability across studies

Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva

Uptake of the Lyme disease spirochete by its tick vector requires not only chemical signals present in the tick’s saliva but a responsive phenotype by the Borrelia burgdorferi living in the mammalian host. This is the principle behind xenodiagnosis, wherein pathogen is detected by vector acquisition. To study migration of B. burgdorferi toward Ixodes scapularis tick saliva, with the goal of identifying chemoattractant molecules, we tested multiple assays and compared migration of host-adapted spirochetes to those cultured in vitro. We tested mammalian host-adapted spirochetes, along with those grown in culture at 34 °C, for their relative attraction to tick saliva or the nutrient N-acetyl-D-glucosamine (D-GlcNAc) and its dimer chitobiose using two different experimental designs. The host-adapted B. burgdorferi showed greater preference for tick saliva over the nutrients, whereas the cultured incubator-grown B. burgdorferi displayed no significant attraction to saliva versus a significant response to the nutrients. Our results not only describe a validated migration assay for studies of the Lyme disease agent, but provide a further understanding of how growth conditions and phenotype of B. burgdorferi are related to vector acquisition

Clinical and Pathological Findings Associated with Aerosol Exposure of Macaques to Ricin Toxin

Ricin is a potential bioweapon that could be used against civilian and military personnel. Aerosol exposure is the most likely route of contact to ricin toxin that will result in the most severe toxicity. Early recognition of ricin exposure is essential if specific antidotes are to be applied. Initial diagnosis will most likely be syndromic, i.e., fitting clinical and laboratory signs into a pattern which then will guide the choice of more specific diagnostic assays and therapeutic interventions. We have studied the pathology of ricin toxin in rhesus macaques exposed to lethal and sublethal ricin aerosols. Animals exposed to lethal ricin aerosols were followed clinically using telemetry, by clinical laboratory analyses and by post-mortem examination. Animals exposed to lethal aerosolized ricin developed fever associated with thermal instability, tachycardia, and dyspnea. In the peripheral blood a marked neutrophilia (without immature bands) developed at 24 h. This was accompanied by an increase in monocytes, but depletion of lymphocytes. Red cell indices indicated hemoconcentration, as did serum chemistries, with modest increases in sodium and blood urea nitrogen (BUN). Serum albumin was strikingly decreased. These observations are consistent with the pathological observations of fluid shifts to the lungs, in the form of hemorrhages, inflammatory exudates, and tissue edema. In macaques exposed to sublethal aerosols of ricin, late pathologic consequences included chronic pulmonary fibrosis, likely mediated by M2 macrophages. Early administration of supportive therapy, specific antidotes after exposure or vaccines prior to exposure have the potential to favorably alter this outcome

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

Nonpathologic Infection of Macaques by an Attenuated Mycobacterial Vaccine Is Not Reactivated in the Setting of HIV Co-Infection

Failure to replace Bacille Calmette-Guerin vaccines with efficacious anti-tuberculosis (TB) vaccines have prompted outside-the-box thinking, including pulmonary vaccination to elicit local immunity. Inhalational MtbΔsigH, a stress-response-attenuated strain, protected against lethal TB in macaques. While live mycobacterial vaccines show promising efficacy, HIV co-infection and the resulting immunodeficiency prompts safety concerns about their use. We assessed the persistence and safety of MtbΔsigH, delivered directly to the lungs, in the setting of HIV co-infection. Macaques were aerosol-vaccinated with ΔsigH and subsequently challenged with SIVmac. Bronchoalveolar lavage and tissues were sampled for mycobacterial persistence, pathology, and immune correlates. Only 35% and 3.5% of lung samples were positive for live bacilli and granulomas, respectively. Our results therefore suggest that the nonpathologic infection of macaque lungs by ΔsigH was not reactivated by simian immunodeficiency virus, despite high viral levels and massive ablation of pulmonary CD4 T cells. Protective pulmonary responses were retained, including vaccine-induced bronchus-associated lymphoid tissue and CD8 effector memory T cells. Despite acute simian immunodeficiency virus infection, all animals remained asymptomatic of pulmonary TB. These findings highlight the efficacy of mucosal vaccination via this attenuated strain and will guide its further development to potentially combat TB in HIV-endemic areas. Our results also suggest that a lack of pulmonary pathology is a key correlate of the safety of live mycobacterial vaccines