Protective effect of pre-existing natural immunity in a nonhuman primate reinfection model of congenital cytomegalovirus infection.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of neurologic defects in newborns with particularly severe sequelae in the setting of primary CMV infection in the first trimester of pregnancy. The majority of cCMV cases worldwide occur after non-primary infection in CMV-seropositive women; yet the extent to which pre-existing natural CMV-specific immunity protects against CMV reinfection or reactivation during pregnancy remains ill-defined. We previously reported on a novel nonhuman primate model of cCMV in rhesus macaques where 100% placental transmission and 83% fetal loss were seen in CD4+ T lymphocyte-depleted rhesus CMV (RhCMV)-seronegative dams after primary RhCMV infection. To investigate the protective effect of preconception maternal immunity, we performed reinfection studies in CD4+ T lymphocyte-depleted RhCMV-seropositive dams inoculated in late first / early second trimester gestation with RhCMV strains 180.92 (n = 2), or RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag, a wild-type-like RhCMV clone with SIVgag inserted as an immunological marker, administered separately (n = 3). An early transient increase in circulating monocytes followed by boosting of the pre-existing RhCMV-specific CD8+ T lymphocyte and antibody response was observed in the reinfected dams but not in control CD4+ T lymphocyte-depleted dams. Emergence of SIV Gag-specific CD8+ T lymphocyte responses in macaques inoculated with the FL-RhCMVΔRh13.1/SIVgag virus confirmed reinfection. Placental transmission was detected in only one of five reinfected dams and there were no adverse fetal sequelae. Viral whole genome, short-read, deep sequencing analysis confirmed transmission of both reinfection RhCMV strains across the placenta with ~30% corresponding to FL-RhCMVΔRh13.1/SIVgag and ~70% to RhCMV UCD52, consistent with the mixed human CMV infections reported in infants with cCMV. Our data showing reduced placental transmission and absence of fetal loss after non-primary as opposed to primary infection in CD4+ T lymphocyte-depleted dams indicates that preconception maternal CMV-specific CD8+ T lymphocyte and/or humoral immunity can protect against cCMV infection.

Enhanced complement activation and MAC formation accelerates severe COVID-19.

Emerging evidence indicates that activation of complement system leading to the formation of the membrane attack complex (MAC) plays a detrimental role in COVID-19. However, their pathogenic roles have never been experimentally investigated before. We used three knock out mice strains (1. C3-/-; 2. C7-/-; and 3. Cd59ab-/-) to evaluate the role of complement in severe COVID-19 pathogenesis. C3 deficient mice lack a key common component of all three complement activation pathways and are unable to generate C3 and C5 convertases. C7 deficient mice lack a complement protein needed for MAC formation. Cd59ab deficient mice lack an important inhibitor of MAC formation. We also used anti-C5 antibody to block and evaluate the therapeutic potential of inhibiting MAC formation. We demonstrate that inhibition of complement activation (in C3-/-) and MAC formation (in C3-/-. C7-/-, and anti-C5 antibody) attenuates severe COVID-19; whereas enhancement of MAC formation (Cd59ab-/-) accelerates severe COVID-19. The degree of MAC but not C3 deposits in the lungs of C3-/-, C7-/- mice, and Cd59ab-/- mice as compared to their control mice is associated with the attenuation or acceleration of SARS-CoV-2-induced disease. Further, the lack of terminal complement activation for the formation of MAC in C7 deficient mice protects endothelial dysfunction, which is associated with the attenuation of diseases and pathologic changes. Our results demonstrated the causative effect of MAC in severe COVID-19 and indicate a potential avenue for modulating the complement system and MAC formation in the treatment of severe COVID-19.

Cerebrospinal Fluid Protein Markers Indicate Neuro-Damage in SARS-CoV-2-Infected Nonhuman Primates.

Neurologic manifestations are among the most frequently reported complications of COVID-19. However, given the paucity of tissue samples and the highly infectious nature of the etiologic agent of COVID-19, we have limited information to understand the neuropathogenesis of COVID-19. Therefore, to better understand the impact of COVID-19 on the brain, we used mass-spectrometry-based proteomics with a data-independent acquisition mode to investigate cerebrospinal fluid (CSF) proteins collected from two different nonhuman primates, Rhesus Macaque and African Green Monkeys, for the neurologic effects of the infection. These monkeys exhibited minimal to mild pulmonary pathology but moderate to severe central nervous system (CNS) pathology. Our results indicated that CSF proteome changes after infection resolution corresponded with bronchial virus abundance during early infection and revealed substantial differences between the infected nonhuman primates and their age-matched uninfected controls, suggesting these differences could reflect altered secretion of CNS factors in response to SARS-CoV-2-induced neuropathology. We also observed the infected animals exhibited highly scattered data distributions compared to their corresponding controls indicating the heterogeneity of the CSF proteome change and the host response to the viral infection. Dysregulated CSF proteins were preferentially enriched in functional pathways associated with progressive neurodegenerative disorders, hemostasis, and innate immune responses that could influence neuroinflammatory responses following COVID-19. Mapping these dysregulated proteins to the Human Brain Protein Atlas found that they tended to be enriched in brain regions that exhibit more frequent injury following COVID-19. It, therefore, appears reasonable to speculate that such CSF protein changes could serve as signatures for neurologic injury, identify important regulatory pathways in this process, and potentially reveal therapeutic targets to prevent or attenuate the development of neurologic injuries following COVID-19.

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.

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.

The pigtail macaque (Macaca nemestrina) model of COVID-19 reproduces diverse clinical outcomes and reveals new and complex signatures of disease.

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over five million people worldwide as of December 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics.

Elevated soluble urokinase plasminogen activator receptor is associated with renal dysfunction in a Chlorocebus atheiops COVID-19 model.

Increases of soluble urokinase plasminogen activator receptor (suPAR) were measured in both urine and plasma of a Chlorocebus aethiops (African green monkey; AGM) mucosal infected with SARS-CoV-2. The data indicate that elevated suPAR may be associated with renal dysfunction and pathology in the context of COVID-19.

Breakthrough Gastrointestinal COVID-19 and Intrahost Evolution Consequent to Combination Monoclonal Antibody Prophylaxis.

Breakthrough gastrointestinal COVID-19 was observed after experimental SARS-CoV-2 upper mucosal infection in a rhesus macaque undergoing low-dose monoclonal antibody prophylaxis. High levels of viral RNA were detected in intestinal sites contrasting with minimal viral replication in upper respiratory mucosa. Sequencing of virus recovered from tissue in 3 gastrointestinal sites and rectal swab revealed loss of furin cleavage site deletions present in the inoculating virus stock and 2 amino acid changes in spike that were detected in 2 colon sites but not elsewhere, suggesting compartmentalized replication and intestinal viral evolution. This suggests suboptimal antiviral therapies promote viral sequestration in these anatomies.

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.

Immunopathogenesis in HIV-associated pediatric tuberculosis.

Tuberculosis (TB) is an increasing global emergency in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, in which host immunity is dysregulated and compromised. However, the pathogenesis and efficacy of therapeutic strategies in HIV-associated TB in developing infants are essentially lacking. Bacillus Calmette-Guerin vaccine, an attenuated live strain of Mycobacterium bovis, is not adequately effective, which confers partial protection against Mycobacterium tuberculosis (Mtb) in infants when administered at birth. However, pediatric HIV infection is most devastating in the disease progression of TB. It remains challenging whether early antiretroviral therapy (ART) could maintain immune development and function, and restore Mtb-specific immune function in HIV-associated TB in children. A better understanding of the immunopathogenesis in HIV-associated pediatric Mtb infection is essential to provide more effective interventions, reducing the risk of morbidity and mortality in HIV-associated Mtb infection in infants. IMPACT: Children living with HIV are more likely prone to opportunistic infection, predisposing high risk of TB diseases. HIV and Mtb coinfection in infants may synergistically accelerate disease progression. Early ART may probably induce immune reconstitution inflammatory syndrome and TB pathology in HIV/Mtb coinfected infants.

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.

Comparative Analysis of Gammaherpesvirus Circular RNA Repertoires: Conserved and Unique Viral Circular RNAs.

Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication.IMPORTANCE Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.

Elite Control, Gut CD4 T Cell Sparing, and Enhanced Mucosal T Cell Responses in Macaca nemestrina Infected by a Simian Immunodeficiency Virus Lacking a gp41 Trafficking Motif.

UNLABELLED: Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques (Macaca mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4(+) T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques (Macaca nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4(+) T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of <15 to 50 RNA copies/ml. CD4(+) T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4(+) T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8(+) cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE: The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

Deficiency of Tlr7 and Irf7 in mice increases the severity of COVID-19 through the reduced interferon production.

Toll-like receptor 7 (Tlr7) deficiency-accelerated severe COVID-19 is associated with reduced production of interferons (IFNs). However, the underlying mechanisms remain elusive. To address these questions, we utilize Tlr7 and Irf7 deficiency mice, single-cell RNA analysis together with bone marrow transplantation approaches. We demonstrate that at the early phase of infection, SARS-CoV-2 causes the upregulation of Tlr7, Irf7, and IFN pathways in the lungs of the infected mice. The deficiency of Tlr7 and Irf7 globally and/or in immune cells in mice increases the severity of COVID-19 via impaired IFN activation in both immune and/or non-immune cells, leading to increased lung viral loads. These effects are associated with reduced IFN alpha and gamma levels in the circulation. The deficiency of Tlr7 tends to cause the reduced production and nuclear translocation of interferon regulatory factor 7 (IRF7) in the lungs of the infected mice, indicative of reduced IRF7 activation. Despite higher amounts of lung viral antigen, Tlr7 or Irf7 deficiency resulted in substantially reduced production of antibodies against SARS-CoV-2, thereby delaying the viral clearance. These results highlight the importance of the activation of TLR7 and IRF7 leading to IFN production on the development of innate and adaptive immunity against COVID-19.

Natural Killer Cells Do Not Attenuate a Mouse-Adapted SARS-CoV-2-Induced Disease in Rag2-/- Mice.

This study investigates the roles of T, B, and Natural Killer (NK) cells in the pathogenesis of severe COVID-19, utilizing mouse-adapted SARS-CoV-2-MA30 (MA30). To evaluate this MA30 mouse model, we characterized MA30-infected C57BL/6 mice (B6) and compared them with SARS-CoV-2-WA1 (an original SARS-CoV-2 strain) infected K18-human ACE2 (K18-hACE2) mice. We found that the infected B6 mice developed severe peribronchial inflammation and rapid severe pulmonary edema, but less lung interstitial inflammation than the infected K18-hACE2 mice. These pathological findings recapitulate some pathological changes seen in severe COVID-19 patients. Using this MA30-infected mouse model, we further demonstrate that T and/or B cells are essential in mounting an effective immune response against SARS-CoV-2. This was evident as Rag2-/- showed heightened vulnerability to infection and inhibited viral clearance. Conversely, the depletion of NK cells did not significantly alter the disease course in Rag2-/- mice, underscoring the minimal role of NK cells in the acute phase of MA30-induced disease. Together, our results indicate that T and/or B cells, but not NK cells, mitigate MA30-induced disease in mice and the infected mouse model can be used for dissecting the pathogenesis and immunology of severe COVID-19.

MVA-based vaccines are protective against lethal eastern equine encephalitis virus aerosol challenge in cynomolgus macaques.

MVA-based monovalent eastern equine encephalitis virus (MVA-BN-EEEV) and multivalent western, eastern, and Venezuelan equine encephalitis virus (MVA-BN-WEV) vaccines were evaluated in the cynomolgus macaque aerosol model of EEEV infection. Macaques vaccinated with two doses of 5 × 108 infectious units of the MVA-BN-EEEV or MVA-BN-WEV vaccine by the intramuscular route rapidly developed robust levels of neutralizing antibodies to EEEV that persisted at high levels until challenge at day 84 via small particle aerosol delivery with a target inhaled dose of 107 PFU of EEEV FL93-939. Robust protection was observed, with 7/8 animals receiving MVA-BN-EEEV and 100% (8/8) animals receiving MVA-BN-WEV surviving while only 2/8 mock vaccinated controls survived lethal challenge. Complete protection from viremia was afforded by both vaccines, with near complete protection from vRNA loads in tissues and any pathologic evidence of central nervous system damage. Overall, the results indicate both vaccines are effective in eliciting an immune response that is consistent with protection from aerosolized EEEV-induced disease.

Elevated Inflammation Associated with Markers of Neutrophil Function and Gastrointestinal Disruption in Pilot Study of Plasmodium fragile Co-Infection of ART-Treated SIVmac239+ Rhesus Macaques.

Human immunodeficiency virus (HIV) and malaria, caused by infection with Plasmodium spp., are endemic in similar geographical locations. As a result, there is high potential for HIV/Plasmodium co-infection, which increases the pathology of both diseases. However, the immunological mechanisms underlying the exacerbated disease pathology observed in co-infected individuals are poorly understood. Moreover, there is limited data available on the impact of Plasmodium co-infection on antiretroviral (ART)-treated HIV infection. Here, we used the rhesus macaque (RM) model to conduct a pilot study to establish a model of Plasmodium fragile co-infection during ART-treated simian immunodeficiency virus (SIV) infection, and to begin to characterize the immunopathogenic effect of co-infection in the context of ART. We observed that P. fragile co-infection resulted in parasitemia and anemia, as well as persistently detectable viral loads (VLs) and decreased absolute CD4+ T-cell counts despite daily ART treatment. Notably, P. fragile co-infection was associated with increased levels of inflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1). P. fragile co-infection was also associated with increased levels of neutrophil elastase, a plasma marker of neutrophil extracellular trap (NET) formation, but significant decreases in markers of neutrophil degranulation, potentially indicating a shift in the neutrophil functionality during co-infection. Finally, we characterized the levels of plasma markers of gastrointestinal (GI) barrier permeability and microbial translocation and observed significant correlations between indicators of GI dysfunction, clinical markers of SIV and Plasmodium infection, and neutrophil frequency and function. Taken together, these pilot data verify the utility of using the RM model to examine ART-treated SIV/P. fragile co-infection, and indicate that neutrophil-driven inflammation and GI dysfunction may underlie heightened SIV/P. fragile co-infection pathogenesis.

The Impact of SIV-Induced Immunodeficiency on SARS-CoV-2 Disease, Viral Dynamics, and Antiviral Immune Response in a Nonhuman Primate Model of Coinfection.

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. In this pilot study, we exposed two pigtail macaques (PTMs) chronically infected with SIVmac239, exhibiting from very low to no CD4 T cells across all compartments, to SARS-CoV-2. We monitored the disease progression, viral replication, and evolution, and compared these outcomes with SIV-naïve PTMs infected with SARS-CoV-2. No overt signs of COVID-19 disease were observed in either animal, and the SARS-CoV-2 viral kinetics and evolution in the SIVmac239 PTMs were indistinguishable from those in the SIV-naïve PTMs in all sampled mucosal sites. However, the single-cell RNA sequencing of bronchoalveolar lavage cells revealed an infiltration of functionally inert monocytes after SARS-CoV-2 infection. Critically, neither of the SIV-infected PTMs mounted detectable anti-SARS-CoV-2 T-cell responses nor anti-SARS-CoV-2 binding or neutralizing antibodies. Thus, HIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants but may remove the ability of infected individuals to mount adaptive immune responses against SARS-CoV-2.

Mid-titer human convalescent plasma administration results in suboptimal prophylaxis against SARS-CoV-2 infection in rhesus macaques.

Introduction: ARS-CoV-2 is a respiratory pathogen currently causing a worldwide pandemic, with resulting pathology of differing severity in humans, from mild illness to severe disease and death. The rhesus macaque model of COVID-19 was utilized to evaluate the added benefit of prophylactic administration of human post-SARS-CoV-2 infection convalescent plasma (CP) on disease progression and severity. Methods: A pharmacokinetic (PK) study using CP in rhesus monkeys preceded the challenge study and revealed the optimal time of tissue distribution for maximal effect. Thereafter, CP was administered prophylactically three days prior to mucosal SARS-CoV-2 viral challenge. Results: Results show similar viral kinetics in mucosal sites over the course of infection independent of administration of CP or normal plasma, or historic controls with no plasma. No changes were noted upon necropsy via histopathology, although there were differences in levels of vRNA in tissues, with both normal and CP seemingly blunting viral loads. Discussion: Results indicate that prophylactic administration with mid-titer CP is not effective in reducing disease severity of SARS-CoV-2 infection in the rhesus COVID-19 disease model.

COVID-19 and influenza infections mediate distinct pulmonary cellular and transcriptomic changes.

SARS-CoV-2 infection can cause persistent respiratory sequelae. However, the underlying mechanisms remain unclear. Here we report that sub-lethally infected K18-human ACE2 mice show patchy pneumonia associated with histiocytic inflammation and collagen deposition at 21 and 45 days post infection (DPI). Transcriptomic analyses revealed that compared to influenza-infected mice, SARS-CoV-2-infected mice had reduced interferon-gamma/alpha responses at 4 DPI and failed to induce keratin 5 (Krt5) at 6 DPI in lung, a marker of nascent pulmonary progenitor cells. Histologically, influenza- but not SARS-CoV-2-infected mice showed extensive Krt5+ "pods" structure co-stained with stem cell markers Trp63/NGFR proliferated in the pulmonary consolidation area at both 7 and 14 DPI, with regression at 21 DPI. These Krt5+ "pods" structures were not observed in the lungs of SARS-CoV-2-infected humans or nonhuman primates. These results suggest that SARS-CoV-2 infection fails to induce nascent Krt5+ cell proliferation in consolidated regions, leading to incomplete repair of the injured lung.