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.

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.

The dynamics of γδ T cell responses in nonhuman primates during SARS-CoV-2 infection.

Although most SARS-CoV-2 infections are mild, some patients develop systemic inflammation and progress to acute respiratory distress syndrome (ARDS). However, the cellular mechanisms underlying this spectrum of disease remain unclear. γδT cells are T lymphocyte subsets that have key roles in systemic and mucosal immune responses during infection and inflammation. Here we show that peripheral γδT cells are rapidly activated following aerosol or intra-tracheal/intra-nasal (IT/IN) SARS-CoV-2 infection in nonhuman primates. Our results demonstrate a rapid expansion of Vδ1 γδT cells at day1 that correlate significantly with lung viral loads during the first week of infection. Furthermore, increase in levels of CCR6 and Granzyme B expression in Vδ1 T cells during viral clearance imply a role in innate-like epithelial barrier-protective and cytotoxic functions. Importantly, the early activation and mobilization of circulating HLA-DR+CXCR3+ γδT cells along with significant correlations of Vδ1 T cells with IL-1Ra and SCF levels in bronchoalveolar lavage suggest a novel role for Vδ1 T cells in regulating lung inflammation during aerosol SARS-CoV-2 infection. A deeper understanding of the immunoregulatory functions of MHC-unrestricted Vδ1 T cells in lungs during early SARS-CoV-2 infection is particularly important in the wake of emerging new variants with increased transmissibility and immune evasion potential.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Dose, Infection, and Disease Outcomes for Coronavirus Disease 2019 (COVID-19): A Review.

The relationship between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dose, infection, and coronavirus disease 2019 (COVID-19) outcomes remains poorly understood. This review summarizes the existing literature regarding this issue, identifies gaps in current knowledge, and suggests opportunities for future research. In humans, host characteristics, including age, sex, comorbidities, smoking, and pregnancy, are associated with severe COVID-19. Similarly, in animals, host factors are strong determinants of disease severity, although most animal infection models manifest clinically with mild to moderate respiratory disease. The influence of variants of concern as it relates to infectious dose, consequence of overall pathogenicity, and disease outcome in dose-response remains unknown. Epidemiologic data suggest a dose-response relationship for infection contrasting with limited and inconsistent surrogate-based evidence between dose and disease severity. Recommendations include the design of future infection studies in animal models to investigate inoculating dose on outcomes and the use of better proxies for dose in human epidemiology studies.

SARS-CoV-2 Epitopes following Infection and Vaccination Overlap Known Neutralizing Antibody Sites.

Identification of epitopes targeted following virus infection or vaccination can guide vaccine design and development of therapeutic interventions targeting functional sites, but can be laborious. Herein, we employed peptide microarrays to map linear peptide epitopes (LPEs) recognized following SARS-CoV-2 infection and vaccination. LPEs detected by nonhuman primate (NHP) and patient IgMs after SARS-CoV-2 infection extensively overlapped, localized to functionally important virus regions, and aligned with reported neutralizing antibody binding sites. Similar LPE overlap occurred after infection and vaccination, with LPE clusters specific to each stimulus, where strong and conserved LPEs mapping to sites known or likely to inhibit spike protein function. Vaccine-specific LPEs tended to map to sites known or likely to be affected by structural changes induced by the proline substitutions in the mRNA vaccine's S protein. Mapping LPEs to regions of known functional importance in this manner may accelerate vaccine evaluation and discovery of targets for site-specific therapeutic interventions.

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.

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.

Bioaerosols and airborne transmission: Integrating biological complexity into our perspective.

There is broad consensus that airborne disease transmission continues to be the thematic focus of COVID-19, the complexities and understanding of which continues to complicate our attempts to control this pandemic. Masking used as both personal protection and source reduction predominates our society at present and, other than vaccination, remains the public health measure that will faithfully reduce aerosol transmission and overall disease burden (Gandhi and Marr, 2021). Early in the advent of the COVID-19 pandemic, and especially after preliminary recognition of airborne transmission, there was considerable efforts in the application of computational fluid dynamics (CFD) modeling aerosols as well as risk models calculations, the products of which were detailed in the literature (Morawska et al., 2020; Buonanno et al., 2020a) and even disseminated in media destined for the public. As the respiratory pathway emerged as the dominant exposure pathway for SARSCoV-2 transmission, much of what was promoted from CFD was applied to risk models to estimate community infection and in some cases expected clinical outcome. COVID-19 proved to fit the profile of an obligate respiratory-transmitted pathogen, and the plausibility of using aerosol modeling when silhouetted with emerging COVID-19 epidemiology provided ample evidence for promotion of masking and ventilation optimization as a required public health measure. Masking is often included as a factor in developed risk models and it remains an essentially important part of our response to this airborne threat, and ultimately will agnostically reduce disease burden although efforts to improve ventilation in indoor spaces remain a challenge. Arguably the most important concept in the airborne transmission of infectious agents is the biologically active componentry that comprises the aerosol particle and the functional dynamic nature of particle contents. Specifically, the innate generation, transport, and ultimate deposition/disposition of bioaerosols; the aerosol particles that nearly exclusively harbor bioactive components, including viruses, when disease agents are transmitted through the air.

A Miniaturized Electrostatic Precipitator Respirator Effectively Removes Ambient SARS-CoV-2 Bioaerosols.

The inhalation of ambient SARS-CoV-2-containing bioaerosols leads to infection and pandemic airborne transmission in susceptible populations. Filter-based respirators effectively reduce exposure but complicate normal respiration through breathing zone pressure differentials; therefore, they are impractical for long-term use. OBJECTIVES: We tested the comparative effectiveness of a prototyped miniaturized electrostatic precipitator (mEP) on a filter-based respirator (N95) via the removal of viral bioaerosols from a simulated, inspired air stream. Methods: Each respirator was tested within a 16 L environmental chamber housed within a Class III biological safety cabinet within biosafety level 3 containment. SARS-CoV-2-containing bioaerosols were generated in the chamber, drawn by a vacuum through each respirator, and physical particle removal and viral genomic RNA were measured distal to the breathing zone of each device. MEASUREMENTS AND MAIN RESULTS: The mEP respirator removed particles (96.5 ± 0.4%), approximating efficiencies of the N95 (96.9 ± 0.6%). The mEP respirator similarly decreased SARS-CoV-2 viral RNA (99.792%) when compared to N95 removal (99.942%), as a function of particle removal from the airstream distal to the breathing zone of each respirator. CONCLUSIONS: The mEP respirator approximated the performance of a filter-based N95 respirator for particle removal and viral RNA as a constituent of the SARS-CoV-2 bioaerosols generated for this evaluation. In practice, the mEP respirator could provide equivalent protection from ambient infectious bioaerosols as the N95 respirator without undue pressure drop to the wearer, thereby facilitating its long-term use in an unobstructed breathing configuration.

Neuropathology and virus in brain of SARS-CoV-2 infected non-human primates.

Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren't well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with "long COVID". Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

Advances and gaps in SARS-CoV-2 infection models.

The global response to Coronavirus Disease 2019 (COVID-19) is now facing new challenges such as vaccine inequity and the emergence of SARS-CoV-2 variants of concern (VOCs). Preclinical models of disease, in particular animal models, are essential to investigate VOC pathogenesis, vaccine correlates of protection and postexposure therapies. Here, we provide an update from the World Health Organization (WHO) COVID-19 modeling expert group (WHO-COM) assembled by WHO, regarding advances in preclinical models. In particular, we discuss how animal model research is playing a key role to evaluate VOC virulence, transmission and immune escape, and how animal models are being refined to recapitulate COVID-19 demographic variables such as comorbidities and age.

CRISPR-based assay reveals SARS-CoV-2 RNA dynamic changes and redistribution patterns in non-human primate model.

Mounting evidence indicates that SARS-CoV-2 can infect multiple systemic tissues, but few studies have evaluated SARS-CoV-2 RNA dynamics in multiple specimen types due to their reduced accessibility and diminished performance of RT-qPCR with non-respiratory specimens. Here, we employed an ultrasensitive CRISPR-RT-PCR assay to analyze longitudinal mucosal (nasal, buccal, pharyngeal, and rectal), plasma, and breath samples from SARS-CoV-2-infected non-human primates (NHPs) to detect dynamic changes in SARS-CoV-2 RNA level and distribution among these specimens. We observed that CRISPR-RT-PCR results consistently detected SARS-CoV-2 RNA in all sample types at most time points post-infection, and that SARS-CoV-2 infection dose and administration route did not markedly affect the CRISPR-RT-PCR signal detected in most specimen types. However, consistent RT-qPCR positive results were restricted to nasal, pharyngeal, and rectal swab samples, and tended to decrease earlier than CRISPR-RT-PCR results, reflecting lower assay sensitivity. SARS-CoV-2 RNA was detectable in both pulmonary and extrapulmonary specimens from early to late infection by CRISPR-RT-PCR, albeit with different abundance and kinetics, with SARS-CoV-2 RNA increases detected in plasma and rectal samples trailing those detected in upper respiratory tract samples. CRISPR-RT-PCR assays for SARS-CoV-2 RNA in non-respiratory specimens may thus permit direct diagnosis of suspected COVID-19 cases missed by RT-PCR, while tracking SARS-CoV-2 RNA in minimally invasive alternate specimens may better evaluate the progression and resolution of SARS-CoV-2 infections.

Development of an In Vivo Probe to Track SARS-CoV-2 Infection in Rhesus Macaques

Infection with the novel coronavirus, SARS-CoV-2, results in pneumonia and other respiratory symptoms as well as pathologies at diverse anatomical sites. An outstanding question is whether these diverse pathologies are due to replication of the virus in these anatomical compartments and how and when the virus reaches those sites. To answer these outstanding questions and study the spatiotemporal dynamics of SARS-CoV-2 infection a method for tracking viral spread in vivo is needed. We developed a novel, fluorescently labeled, antibody-based in vivo probe system using the anti-spike monoclonal antibody CR3022 and demonstrated that it could successfully identify sites of SARS-CoV-2 infection in a rhesus macaque model of COVID-19. Our results showed that the fluorescent signal from our antibody-based probe could differentiate whole lungs of macaques infected for 9 days from those infected for 2 or 3 days. Additionally, the probe signal corroborated the frequency and density of infected cells in individual tissue blocks from infected macaques. These results provide proof of concept for the use of in vivo antibody-based probes to study SARS-CoV-2 infection dynamics in rhesus macaques.

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.

Intra-Host SARS-CoV-2 Evolution in the Gut of Mucosally-Infected Chlorocebus aethiops (African Green Monkeys).

In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were not detected in viral RNA recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, which were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future primate models involving SARS-CoV-2 infection.

Effective Prophylaxis of COVID-19 in Rhesus Macaques Using a Combination of Two Parenterally-Administered SARS-CoV-2 Neutralizing Antibodies.

SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.

SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

Liposome-mediated detection of SARS-CoV-2 RNA-positive extracellular vesicles in plasma.

Plasma SARS-CoV-2 RNA may represent a viable diagnostic alternative to respiratory RNA levels, which rapidly decline after infection. Quantitative PCR with reverse transcription (RT-qPCR) reference assays exhibit poor performance with plasma, probably reflecting the dilution and degradation of viral RNA released into the circulation, but these issues could be addressed by analysing viral RNA packaged into extracellular vesicles. Here we describe an assay approach in which extracellular vesicles directly captured from plasma are fused with reagent-loaded liposomes to sensitively amplify and detect a SARS-CoV-2 gene target. This approach accurately identified patients with COVID-19, including challenging cases missed by RT-qPCR. SARS-CoV-2-positive extracellular vesicles were detected at day 1 post-infection, and plateaued from day 6 to the day 28 endpoint in a non-human primate model, while signal durations for 20-60 days were observed in young children. This nanotechnology approach uses a non-infectious sample and extends virus detection windows, offering a tool to support COVID-19 diagnosis in patients without SARS-CoV-2 RNA detectable in the respiratory tract.

A New Natural Defense Against Airborne Pathogens.

We propose the nasal administration of calcium-enriched physiological salts as a new hygienic intervention with possible therapeutic application as a response to the rapid and tenacious spread of COVID-19. We test the effectiveness of these salts against viral and bacterial pathogens in animals and humans. We find that aerosol administration of these salts to the airways diminishes the exhalation of the small particles that face masks fail to filter and, in the case of an influenza swine model, completely block airborne transmission of disease. In a study of 10 human volunteers (5 less than 65 years and 5 older than 65 years), we show that delivery of a nasal saline comprising calcium and sodium salts quickly (within 15 min) and durably (up to at least 6 h) diminishes exhaled particles from the human airways. Being predominantly smaller than 1 µm, these particles are below the size effectively filtered by conventional masks. The suppression of exhaled droplets by the nasal delivery of calcium-rich saline with aerosol droplet size of around 10 µm suggests the upper airways as a primary source of bioaerosol generation. The suppression effect is especially pronounced (99%) among those who exhale large numbers of particles. In our study, we found this high-particle exhalation group to correlate with advanced age. We argue for a new hygienic practice of nasal cleansing by a calcium-rich saline aerosol, to complement the washing of hands with ordinary soap, use of a face mask, and social distancing.

COVID-19: Famotidine, Histamine, Mast Cells, and Mechanisms.

SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. SARS-CoV-2 infection is necessary but not sufficient for development of clinical COVID-19 disease. Currently, there are no approved pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We have investigated several plausible hypotheses for famotidine activity including antiviral and host-mediated mechanisms of action. We propose that the principal mechanism of action of famotidine for relieving COVID-19 symptoms involves on-target histamine receptor H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release. Based on these findings and associated hypothesis, new COVID-19 multi-drug treatment strategies based on repurposing well-characterized drugs are being developed and clinically tested, and many of these drugs are available worldwide in inexpensive generic oral forms suitable for both outpatient and inpatient treatment of COVID-19 disease.