Myeloid cell-driven nonregenerative pulmonary scarring is conserved in multiple nonhuman primate species regardless of SARS-CoV-2 infection modality (preprint)

The novel coronavirus SARS-CoV-2 has caused a worldwide pandemic resulting in widespread efforts in development of animal models that recapitulate human disease for evaluation of medical countermeasures, and to dissect COVID-19 immunopathogenesis. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM) of nonhuman primates. Species-specific cohorts of RM and AGM Rhesus macaques (Macaca mulatta, RMs) and African green monkeys (Chlorocebus aethiops, AGMs) were experimentally infected with homologous SARS-CoV-2 by either direct mucosal instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated equivalent infection initially by either exposure route although the magnitude and duration of viral loading was greater in AGMs than that of the RM. Clinical onset was nearly immediate (+1dpi) in mucosally-exposed cohorts whereas aerosol-infected animals began to show signs +7dpi. Myeloid cell responses indicative of the development of pulmonary scarring and extended lack of regenerative capacity in the pulmonary compartment was a conserved pathologic response in both species by either exposure modality. This pathological commonality may be useful in future anti-fibrosis therapeutic evaluations and expands our understanding of how SARS-CoV-2 infection leads to ARDS and functional lung damage.

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

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over four million people worldwide as of July 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) (preprint)

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 subsequently lost in virus 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, that were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution unique to this nonhuman primate species and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future development of primate disease models.

An Orally Available Cathepsin L Inhibitor Protects Lungs Against SARS-CoV-2-Induced Diffuse Alveolar Damage in African Green Monkeys (preprint)

The COVID-19 pandemic resulted from global infection by the SARS-CoV-2 coronavirus and rapidly emerged as an urgent health issue requiring effective treatments. To initiate infection, the Spike protein of SARS-CoV-2 requires proteolytic processing mediated by host proteases. Among the host proteases proposed to carry out this activation is the cysteine protease cathepsin L. Inhibiting cathepsin L has been proposed as a therapeutic strategy for treating COVID-19. SLV213 (K777) is an orally administered small molecule protease inhibitor that exhibits in vitro activity against a range of viruses, including SARS-CoV-2. To confirm efficacy in vivo, K777 was evaluated in an African green monkey (AGM) model of COVID-19. A pilot experiment was designed to test K777 in a prophylactic setting, animals were pre-treated with 100mg/kg K777 (N=4) or vehicle (N=2) before inoculation with SARS-CoV-2. Initial data demonstrated that K777 treatment reduced pulmonary pathology compared to vehicle-treated animals. A second study was designed to test activity in a therapeutic setting, with K777 treatment (33 mg/kg or 100 mg/kg) initiated 8 hours after exposure to the virus. In both experiments, animals received K777 daily via oral gavage for 7 days. Vehicle-treated animals exhibited higher lung weights, pleuritis, and diffuse alveolar damage. In contrast, lung pathology was reduced in K777-treated monkeys, and histopathological analyses confirmed the lack of diffuse alveolar damage. Antiviral effects were further demonstrated by quantitative reductions in viral load of samples collected from upper and lower airways. These preclinical data support the potential for early SLV213 treatment in COVID-19 patients to prevent severe lung pathology and disease progression.

Effective prophylaxis of COVID-19 in rhesus macaques using a combination of two parentally-administered SARS-CoV-2 neutralizing antibodies (preprint)

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.

Exhaled aerosol increases with COVID-19 infection, and risk factors of disease symptom severity (preprint)

Coronavirus disease-19 (COVID-19) transmits by droplets generated from surfaces of airway mucus during processes of respiration within hosts infected by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) virus. We studied respiratory droplet generation and exhalation in human and nonhuman primate subjects with and without COVID-19 infection to explore whether SARS-CoV-2 infection, and other changes in physiological state, translates into observable evolution of numbers and sizes of exhaled respiratory droplets in healthy and diseased subjects. In our observational cohort study of the exhaled breath particles of 74 healthy human subjects, and in our experimental infection study of eight nonhuman primates infected by aerosol with SARS-CoV-2, we found that exhaled aerosol particles increase one to three orders of magnitude with aging, high BMI, and COVID-19 infection. These variances appear to be related to changes in airway mucus surface composition and the propensity for mucus surfaces to breakup into small droplets during acts of breathing. We also observed that 20% of those participating in our human study accounted for 80% of the overall exhaled bioaerosol, reflecting a bioaerosol distribution analogous to a classical 20:80 super spreader distribution.

Cellular events of acute, resolving or progressive COVID-19 in SARS-CoV-2 infected non-human primates (preprint)

We investigated the immune events following SARS-CoV-2 infection, from the acute inflammatory state up to four weeks post infection, in non-human primates (NHP) with heterogeneous pulmonary pathology. The acute phase was characterized by a robust and rapid migration of monocytes expressing CD16 from the blood and concomitant increase in CD16+ macrophages in the lungs. We identified two subsets of interstitial macrophages (HLA-DR+ CD206-), a transitional CD11c+ CD16+ cell population that was directly associated with IL-6 levels in plasma, and one long lasting CD11b+ CD16+ cell population. Strikingly, levels of monocytes were a correlate of viral replication in bronchial brushes and we discovered TARC (CCL17) as a new potential mediator of myeloid recruitment to the lungs. Worse disease outcomes were associated with high levels of cell infiltration in lungs including CD11b+ CD16hi macrophages and CD11b+ neutrophils. Accumulation of macrophages was long-lasting and detectable even in animals with mild or no signs of disease. Interestingly, animals with anti-inflammatory responses including high IL-10:IL-6 and kynurenine to tryptophan ratios had less signs of disease. Our results unravel cellular mechanisms of COVID-19 and suggest that NHP may be appropriate models to test immune therapies.

ARDS and Cytokine Storm in SARS-CoV-2 Infected Caribbean Vervets (preprint)

SARS-CoV-2 induces a wide range of disease severity ranging from asymptomatic infection, to a life-threating illness, particularly in the elderly and persons with comorbid conditions. Up to now, SARS-CoV-2 has infected more than five million and led to more than 300,000 deaths worldwide. Among those persons with serious COVID-19 disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. SARS-CoV-2-induced ARDS is difficult to treat clinically, and new therapeutic strategies are needed. In order to evaluate such therapeutic strategies, animal models of SARS-CoV-2 infection that manifest severe disease are needed. Here we report fatal ARDS in two African green monkeys (AGMs) infected with SARS-CoV-2 that demonstrated pathological lesions and disease similar to severe COVID-19 in humans. Moreover, we report the observation of cytokine release (cytokine storm) in three of four infected AGMs. All four animals showed increased levels of IL-6 in plasma, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2 infection. Our results suggest the AGM is a useful model to study disease pathogenesis of SARS-CoV-2, and for the evaluation of therapeutic interventions designed to combat serious pulmonary disease associated with this infection.

The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection (preprint)

Many efforts to design and screen therapeutics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) have focused on inhibiting viral cell entry by disrupting ACE2 binding with the SARS-CoV-2 spike protein. This work focuses on inhibiting SARS-CoV-2 entry through a hypothesized 5{beta}1 integrin-based mechanism, and indicates that inhibiting the spike protein interaction with 5{beta}1 integrin (+/- ACE2), and the interaction between 5{beta}1 integrin and ACE2 using a molecule ATN-161 represents a promising approach to treat COVID-19.

Comparative dynamic aerosol efficiencies of three emergent coronaviruses and the unusual persistence of SARS-CoV-2 in aerosol suspensions (preprint)

The emergent coronavirus, designated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a zoonotic pathogen that has demonstrated remarkable transmissibility in the human population and is the etiological agent of a current global pandemic called COVID-19. We measured the dynamic (short-term) aerosol efficiencies of SARS-CoV-2 and compared the efficiencies with two other emerging coronaviruses, SARS-CoV (emerged in 2002) and Middle Eastern respiratory syndrome CoV (MERS-CoV; emerged starting in 2012). We also quantified the long-term persistence of SARS-CoV-2 and its ability to maintain infectivity when suspended in aerosols for up to 16 hours.