Exposure modality influences viral kinetics but not respiratory outcome of COVID-19 in multiple nonhuman primate species.

The novel coronavirus SARS-CoV-2 emerged in late 2019, rapidly reached pandemic status, and has maintained global ubiquity through the emergence of variants of concern. Efforts to develop animal models have mostly fallen short of recapitulating severe disease, diminishing their utility for research focusing on severe disease pathogenesis and life-saving medical countermeasures. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species of nonhuman primates (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM). Species-specific cohorts were experimentally infected with SARS-CoV-2 by either direct mucosal (intratracheal + intranasal) instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated analogous viral loads in all compartments by either exposure route although the magnitude and duration of viral loading was marginally greater in AGMs than RMs. Clinical onset was nearly immediate (+1dpi) in the mucosal exposure cohort whereas clinical signs and cytokine responses in aerosol exposure animals began +7dpi. Pathologies conserved in both species and both exposure modalities include pulmonary myeloid cell influx, development of pleuritis, and extended lack of regenerative capacity in the pulmonary compartment. Demonstration of conserved pulmonary pathology regardless of species and exposure route expands our understanding of how SARS-CoV-2 infection may lead to ARDS and/or functional lung damage and demonstrates the near clinical response of the nonhuman primate model for anti-fibrotic therapeutic evaluation studies.

The FGF/FGFR system in the microglial neuroinflammation with Borrelia burgdorferi: likely intersectionality with other neurological conditions.

BACKGROUND: Lyme neuroborreliosis, caused by the bacterium Borrelia burgdorferi affects both the central and peripheral nervous systems (CNS, PNS). The CNS manifestations, especially at later stages, can mimic/cause many other neurological conditions including psychiatric disorders, dementia, and others, with a likely neuroinflammatory basis. The pathogenic mechanisms associated with Lyme neuroborreliosis, however, are not fully understood. METHODS: In this study, using cultures of primary rhesus microglia, we explored the roles of several fibroblast growth factor receptors (FGFRs) and fibroblast growth factors (FGFs) in neuroinflammation associated with live B. burgdorferi exposure. FGFR specific siRNA and inhibitors, custom antibody arrays, ELISAs, immunofluorescence and microscopy were used to comprehensively analyze the roles of these molecules in microglial neuroinflammation due to B. burgdorferi. RESULTS: FGFR1-3 expressions were upregulated in microglia in response to B. burgdorferi. Inhibition of FGFR 1, 2 and 3 signaling using siRNA and three different inhibitors showed that FGFR signaling is proinflammatory in response to the Lyme disease bacterium. FGFR1 activation also contributed to non-viable B. burgdorferi mediated neuroinflammation. Analysis of the B. burgdorferi conditioned microglial medium by a custom antibody array showed that several FGFs are induced by the live bacterium including FGF6, FGF10 and FGF12, which in turn induce IL-6 and/or CXCL8, indicating a proinflammatory nature. To our knowledge, this is also the first-ever described role for FGF6 and FGF12 in CNS neuroinflammation. FGF23 upregulation, in addition, was observed in response to the Lyme disease bacterium. B. burgdorferi exposure also downregulated many FGFs including FGF 5, 7, 9, 11, 13, 16, 20 and 21. Some of the upregulated FGFs have been implicated in major depressive disorder (MDD) or dementia development, while the downregulated ones have been demonstrated to have protective roles in epilepsy, Parkinson's disease, Alzheimer's disease, spinal cord injury, blood-brain barrier stability, and others. CONCLUSIONS: In this study we show that FGFRs and FGFs are novel inducers of inflammatory mediators in Lyme neuroborreliosis. It is likely that an unresolved, long-term (neuro)-Lyme infection can contribute to the development of other neurologic conditions in susceptible individuals either by augmenting pathogenic FGFs or by suppressing ameliorative FGFs or both.

Acute Respiratory Distress in Aged, SARS-CoV-2-Infected African Green Monkeys but Not Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a wide range of disease severity, ranging from asymptomatic infection to a life-threating illness, particularly in the elderly population and individuals with comorbid conditions. Among individuals with serious coronavirus 2019 (COVID-19) disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19-induced ARDS and evaluate therapeutic strategies. We report two cases of ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that had pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic, and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. Notable increases in circulating cytokines were observed in three of four infected, aged AGMs but not in infected RMs. All the AGMs had increased levels of plasma IL-6 compared with baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2. Together, our results indicate that both RMs and AGMs are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations, including ARDS.

Lung Expression of Human Angiotensin-Converting Enzyme 2 Sensitizes the Mouse to SARS-CoV-2 Infection.

Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines Cxcl9 and Cxcl10, and genes associated with effector T-cell populations including Cd3 g, Cd8a, and Gzmb. Pathway analysis showed that several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the data set, including cytokine-cytokine receptor interaction, the chemokine signaling pathway, the NOD-like receptor signaling pathway, the measles pathway, and the IL-17 signaling pathway. This response is correlative to clinical response in lungs of patients with COVID-19. These results demonstrate that expression of hACE2 via adenovirus delivery system sensitized the mouse to SARS-CoV-2 infection and resulted in the development of a mild COVID-19 phenotype, highlighting the immune and inflammatory host responses to SARS-CoV-2 infection. This rapidly deployable COVID-19 mouse model is useful for preclinical and pathogenesis studies of COVID-19.

Pericytes as novel targets for HIV/SIV infection in the lung.

Antiretroviral therapy in HIV patients has lengthened lifespan but led to an increased risk for secondary comorbidities, such as pulmonary complications characterized by vascular dysfunction. In the lung, PDGFRß+ mesenchymal cells known as pericytes intimately associate with endothelial cells and are key for their survival both structurally and through the secretion of prosurvival factors. We hypothesize that in HIV infection there are functional changes in pericytes that may lead to destabilization of the microvasculature and ultimately to pulmonary abnormalities. Our objective in this study was to determine whether lung pericytes could be directly infected with HIV. We leveraged lung samples from macaque lungs with or without SIV infection and normal human lung for in vitro experiments. Pericytes were isolated based on the marker platelet-derived growth factor receptor-ß (PDGFRß). We determined that lung PDGFRß-positive (PDGFRß+) pericytes from both macaques and humans express CD4, the primary receptor for SIV/HIV, as well as the major coreceptors CXCR4 and CCR5. We found cells positive for both PDGFRß and SIV in lungs from infected macaques. Lung pericytes isolated from these animals also harbored detectable SIV. To confirm relevance to human disease, we demonstrated that human lung pericytes are capable of being productively infected by HIV in vitro, with the time course of infection suggesting development of viral latency. In summary, we show for the first time that SIV/HIV directly infects lung pericytes, implicating these cells as a novel target and potential reservoir for the virus in vivo.

BCL6 BTB-specific inhibition via FX1 treatment reduces Tfh cells and reverses lymphoid follicle hyperplasia in Indian rhesus macaque (Macaca mulatta).

BACKGROUND: The BTB domain of B-cell lymphoma 6 (BCL6) protein was identified as a therapeutic target for B-cell lymphoma. This study compared the pharmacokinetics (PK) of the BCL6 BTB inhibitor (FX1) between mice and macaques, as well as evaluating its lymphoid suppressive effect in uninfected macaques with lymphoid hyperplasia. MATERIALS AND METHODS: Eight uninfected adult Indian rhesus macaques (Macaca mulatta) were used in the study, four animals carrying lymphoid tissue hyperplasia. Plasma FX1 levels were measured by HPLC-MS/MS. Lymph node biopsies were used for H&E and immunohistochemistry staining, as well as mononuclear cell isolation for flow cytometry analysis. RESULTS: Inhibition of the BCL6 BTB domain with FX1 led to a reduction in the frequency of GC, Tfh CD4+ , and Tfh precursor cells, as well as resolving lymphoid hyperplasia, in rhesus macaques. CONCLUSIONS: B-cell lymphoma 6 inhibition may represent a novel strategy to reduce hyperplastic lymphoid B-cell follicles and decrease Tfh cells.

High Turnover of Tissue Macrophages Contributes to Tuberculosis Reactivation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Background: Tuberculosis (TB) and human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) profoundly affect the immune system and synergistically accelerate disease progression. It is believed that CD4+ T-cell depletion by HIV is the major cause of immunodeficiency and reactivation of latent TB. Previous studies demonstrated that blood monocyte turnover concurrent with tissue macrophage death from virus infection better predicted AIDS onset than CD4+ T-cell depletion in macaques infected with simian immunodeficiency virus (SIV). Methods: In this study, we describe the contribution of macrophages to the pathogenesis of Mycobacterium tuberculosis (Mtb)/SIV coinfection in a rhesus macaque model using in vivo BrdU labeling, immunostaining, flow cytometry, and confocal microscopy. Results: We found that increased monocyte and macrophage turnover and levels of SIV-infected lung macrophages correlated with TB reactivation. All Mtb/SIV-coinfected monkeys exhibited declines in CD4+ T cells regardless of reactivation or latency outcomes, negating lower CD4+ T-cell levels as a primary cause of Mtb reactivation. Conclusions: Results suggest that SIV-related damage to macrophages contributes to Mtb reactivation during coinfection. This also supports strategies to target lung macrophages for the treatment of TB.

Preferential Destruction of Interstitial Macrophages over Alveolar Macrophages as a Cause of Pulmonary Disease in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

To our knowledge, this study demonstrates for the first time that the AIDS virus differentially impacts two distinct subsets of lung macrophages. The predominant macrophages harvested by bronchoalveolar lavage (BAL), alveolar macrophages (AMs), are routinely used in studies on human lung macrophages, are long-lived cells, and exhibit low turnover. Interstitial macrophages (IMs) inhabit the lung tissue, are not recovered with BAL, are shorter-lived, and exhibit higher baseline turnover rates distinct from AMs. We examined the effects of SIV infection on AMs in BAL fluid and IMs in lung tissue of rhesus macaques. SIV infection produced massive cell death of IMs that contributed to lung tissue damage. Conversely, SIV infection induced minimal cell death of AMs, and these cells maintained the lower turnover rate throughout the duration of infection. This indicates that SIV produces lung tissue damage through destruction of IMs, whereas the longer-lived AMs may serve as a virus reservoir to facilitate HIV persistence.

SIV encephalitis lesions are composed of CD163(+) macrophages present in the central nervous system during early SIV infection and SIV-positive macrophages recruited terminally with AIDS.

Macrophage recruitment to the central nervous system (CNS) during AIDS pathogenesis is poorly understood. We measured the accumulation of brain perivascular (CD163(+)) and inflammatory (MAC387(+)) macrophages in SIV-infected monkeys. Monocyte progenitors were 5-bromo-2'-deoxyuridine (BrdU) labeled in bone marrow, and CNS macrophages were labeled serially with fluorescent dextrans injected into the cisterna magna. MAC387(+) macrophages accumulated in the meninges and choroid plexus in early inflammation and in the perivascular space and SIV encephalitis (SIVE) lesions late. CD163(+) macrophages accumulated in the perivascular space and SIVE lesions with late inflammation. Most of the BrdU(+) cells were MAC387(+); however, CD163(+)BrdU(+) macrophages were present in the meninges and choroid plexus with AIDS. Most (81.6% ± 1.8%) of macrophages in SIVE lesions were present in the CNS before SIVE lesion formation. There was a 2.9-fold increase in SIVp28(+) macrophages entering the CNS late compared with those entering early (P < 0.05). The rate of CD163(+) macrophage recruitment to the CNS inversely correlated with time to death (P < 0.03) and increased with SIVE. In SIVE animals, soluble CD163 correlated with CD163(+) macrophage recruitment (P = 0.02). Most perivascular macrophages that comprise SIVE lesions and multinucleated giant cells are present in the CNS early, before SIVE lesions are formed. Most SIV-infected macrophages traffic to the CNS terminally with AIDS.

DosS Is required for the complete virulence of mycobacterium tuberculosis in mice with classical granulomatous lesions.

Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.

Tetherin upregulation in simian immunodeficiency virus-infected macaques.

Here we show that simian immunodeficiency virus (SIV) infection of rhesus macaques results in rapid upregulation of tetherin (BST-2 or CD317) on peripheral blood lymphocytes, including the CD4(+) CCR5(+) T cell targets of virus infection, with a peak of induction that coincides with peak alpha interferon (IFN-α) levels in plasma, and that tetherin remains above baseline levels throughout chronic infection. These observations are consistent with a role for tetherin in innate immunity to immunodeficiency virus infection.

Loss of a tyrosine-dependent trafficking motif in the simian immunodeficiency virus envelope cytoplasmic tail spares mucosal CD4 cells but does not prevent disease progression.

A hallmark of pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections is the rapid and near-complete depletion of mucosal CD4(+) T lymphocytes from the gastrointestinal tract. Loss of these cells and disruption of epithelial barrier function are associated with microbial translocation, which has been proposed to drive chronic systemic immune activation and disease progression. Here, we evaluate in rhesus macaques a novel attenuated variant of pathogenic SIVmac239, termed ΔGY, which contains a deletion of a Tyr and a proximal Gly from a highly conserved YxxØ trafficking motif in the envelope cytoplasmic tail. Compared to SIVmac239, ΔGY established a comparable acute peak of viremia but only transiently infected lamina propria and caused little or no acute depletion of mucosal CD4(+) T cells and no detectable microbial translocation. Nonetheless, these animals developed T-cell activation and declining peripheral blood CD4(+) T cells and ultimately progressed with clinical or pathological features of AIDS. ΔGY-infected animals also showed no infection of macrophages or central nervous system tissues even in late-stage disease. Although the ΔGY mutation persisted, novel mutations evolved, including the formation of new YxxØ motifs in two of four animals. These findings indicate that disruption of this trafficking motif by the ΔGY mutation leads to a striking alteration in anatomic distribution of virus with sparing of lamina propria and a lack of microbial translocation. Because these animals exhibited wild-type levels of acute viremia and immune activation, our findings indicate that these pathological events are dissociable and that immune activation unrelated to gut damage can be sufficient for the development of AIDS.

Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct.

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8α hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8α hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8α TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.

Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct.

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8α hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8α hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8α TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.

Efficient transmission and persistence of low-frequency SIVmac251 variants in CD8-depleted rhesus macaques with different neuropathology.

Infection of CD8-depleted rhesus macaques with the genetically heterogeneous simian immunodeficiency virus (SIV)mac251 viral swarm provides a rapid-disease model for simian acquired immune deficiency syndrome and SIV-encephalitis (SIVE). The objective was to evaluate how the diversity of the swarm influences the initial seeding of the infection that may potentially affect disease progression. Plasma, lymphoid and non-lymphoid (brain and lung) tissues were collected from two infected macaques euthanized at 21 days post-infection (p.i.), as well as longitudinal specimens and post-mortem tissues from four macaques followed throughout the infection. About 1300 gp120 viral sequences were obtained from the infecting SIVmac251 swarm and the macaques longitudinal and post-mortem samples. Phylogenetic and amino acid signature pattern analyses were carried out to assess frequency, transmission dynamics and persistence of specific viral clusters. Although no significant reduction in viral heterogeneity was found early in infection (21 days p.i.), transmission and replication of SIV variants was not entirely random. In particular, two distinct motifs under-represented (<4 %) in the infecting swarm were found at high frequencies (up to 14 %) in all six macaques as early as 21 days p.i. Moreover, a macrophage tropic variant not detected in the viral swarm (<0.3 %) was present at high frequency (29-100 %) in sequences derived from the brain of two macaques with meningitis or severe SIVE. This study demonstrates the highly efficient transmission and persistence in vivo of multiple low frequency SIVmac251 founder variants, characterized by specific gp120 motifs that may be linked to pathogenesis in the rapid-disease model of neuroAIDS.

Functional Insulinomas in a Rhesus Macaque (Macaca mulatta).

We report a rare case of functional insulinomas in a 16.7-year-old female Rhesus macaque (Macaca mulatta) that was presented with neuroglycopenic signs to the breeding colony hospital at the Tulane National Primate Research Center. At initial and follow-up examinations, the animal was consistently hypoglycaemic and was clinically maintained with additional fruits, other high-sugar food items and dextrose supplementation. Occasional episodes of seizure and collapse resolved quickly on administration of high-sugar food items. At necropsy, the uncinate process of the pancreas had a 2.2 cm diameter, red, round, firm neoplastic mass, and another neoplasm was identified on histological examination of the head of pancreas. Histologically, neoplastic cells exhibited neuroendocrine packeting, resembled pancreatic islet cells and immunolabelled for chromogranin A, synaptophysin and insulin but not for somatostatin, gastrin or pancreatic polypeptide. A few cells immunolabelled for glucagon. The clinical signs and gross and histological findings were consistent with functional insulinomas.

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab')2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab')2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.

BACKGROUND: Tuberculosis (TB) and AIDS together present a devastating public health challenge. Over 3 million deaths every year are attributed to these twin epidemics. Annually, ∼11 million people are coinfected with HIV and Mycobacterium tuberculosis (Mtb). AIDS is thought to alter the spontaneous rate of latent TB reactivation. METHODOLOGY: Macaques are excellent models of both TB and AIDS. Therefore, it is conceivable that they can also be used to model coinfection. Using clinical, pathological, and microbiological data, we addressed whether latent TB infection in rhesus macaques can be reactivated by infection with simian immunodeficiency virus (SIV). RESULTS: A low-dose aerosol infection of rhesus macaques with Mtb caused latent, asymptomatic TB infection. Infection of macaques exhibiting latent TB with a rhesus-specific strain of SIV significantly reactivated TB. CONCLUSIONS: Rhesus macaques are excellent model of TB/AIDS coinfection and can be used to study the phenomena of TB latency and reactivation.

Clinicopathologic features of a feline SARS-CoV-2 infection model parallel acute COVID-19 in humans.

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. This study validates a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with severe COVID-19 in humans. Intra-tracheal inoculation of concentrated SARS-CoV-2 caused infected cats to develop clinical disease consistent with that observed in the early exudative phase of COVID-19. A novel clinical scoring system for feline respiratory disease was developed and utilized, documenting a significant degree of lethargy, fever, dyspnea, and dry cough in infected cats. In addition, histopathologic pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were observed due to SARS-CoV-2 infection, imitating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation exists between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were quantified in nasal turbinates, distal trachea, lung, and various other organs. Natural ACE2 expression, paired with clinicopathologic correlates between this feline model and human COVID-19, encourage use of this model for future translational studies.