Inactivation of Ebola Virus and SARS-CoV-2 in Cell Culture Supernatants and Cell Pellets by Gamma Irradiation.

Viral pathogens with the potential to cause widespread disruption to human health and society continue to emerge or re-emerge around the world. Research on such viruses often involves high biocontainment laboratories (BSL3 or BSL4), but the development of diagnostics, vaccines and therapeutics often uses assays that are best performed at lower biocontainment. Reliable inactivation is necessary to allow removal of materials to these spaces and to ensure personnel safety. Here, we validate the use of gamma irradiation to inactivate culture supernatants and pellets of cells infected with a representative member of the Filovirus and Coronavirus families. We show that supernatants and cell pellets containing SARS-CoV-2 are readily inactivated with 1.9 MRad, while Ebola virus requires higher doses of 2.6 MRad for supernatants and 3.8 MRad for pellets. While these doses of radiation inactivate viruses, proinflammatory cytokines that are common markers of virus infection are still detected with low losses. The doses required for virus inactivation of supernatants are in line with previously reported values, but the inactivation of cell pellets has not been previously reported and enables new approaches for analysis of protein-based host responses to infection.

Anti-SARS-CoV-2 activity of targeted kinase inhibitors: Repurposing clinically available drugs for COVID-19 therapy.

Coronavirus disease 2019 (COVID-19) remains a major public health concern, and vaccine unavailability, hesitancy, or failure underscore the need for discovery of efficacious antiviral drug therapies. Numerous approved drugs target protein kinases associated with viral life cycle and symptoms of infection. Repurposing of kinase inhibitors is appealing as they have been vetted for safety and are more accessible for COVID-19 treatment. However, an understanding of drug mechanism is needed to improve our understanding of the factors involved in pathogenesis. We tested the in vitro activity of three kinase inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including inhibitors of AXL kinase, a host cell factor that contributes to successful SARS-CoV-2 infection. Using multiple cell-based assays and approaches, gilteritinib, nintedanib, and imatinib were thoroughly evaluated for activity against SARS-CoV-2 variants. Each drug exhibited antiviral activity, but with stark differences in potency, suggesting differences in host dependency for kinase targets. Importantly, for gilteritinib, the amount of compound needed to achieve 90% infection inhibition, at least in part involving blockade of spike protein-mediated viral entry and at concentrations not inducing phospholipidosis (PLD), approached a clinically achievable concentration. Knockout of AXL, a target of gilteritinib and nintedanib, impaired SARS-CoV-2 variant infectivity, supporting a role for AXL in SARS-CoV-2 infection and supporting further investigation of drug-mediated AXL inhibition as a COVID-19 treatment. This study supports further evaluation of AXL-targeting kinase inhibitors as potential antiviral agents and treatments for COVID-19. Additional mechanistic studies are needed to determine underlying differences in virus response.

Brequinar and dipyridamole in combination exhibits synergistic antiviral activity against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and the associated global pandemic resulting in >400 million infections worldwide and several million deaths. The continued evolution of SARS-CoV-2 to potentially evade vaccines and monoclonal antibody (mAb)-based therapies and the limited number of authorized small-molecule antivirals necessitates the need for development of new drug treatments. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

Identification of potent inhibitors of SARS-CoV-2 infection by combined pharmacological evaluation and cellular network prioritization.

Pharmacologically active compounds with known biological targets were evaluated for inhibition of SARS-CoV-2 infection in cell and tissue models to help identify potent classes of active small molecules and to better understand host-virus interactions. We evaluated 6,710 clinical and preclinical compounds targeting 2,183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target, and cell interactome produced cellular networks important for infection. This analysis revealed 389 small molecules with micromolar to low nanomolar activities, representing >12 scaffold classes and 813 host targets. Representatives were evaluated for mechanism of action in stable and primary human cell models with SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of host factor dependencies and treatments for viral diseases.

Monoclonal antibodies binding data for SARS-CoV-2 proteins.

SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.