Abstract:Highlights d Highly infectious recombinant VSV expressing SARS-CoV-2 spike (S) was generated d rVSV-SARS-CoV-2 S resembles SARS-CoV-2 in entry and inhibitor or antibody sensitivity d rVSV-SARS-CoV-2 S affords rapid screens and forwardgenetic analyses of antivirals Authors
“…A number of surrogate assays that aim to identify molecules with activity against proteins encoded by SARS-CoV-2 have recently been reported [37][38][39] . These assays were developed to provide (Table 1).…”
We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.
“…A number of surrogate assays that aim to identify molecules with activity against proteins encoded by SARS-CoV-2 have recently been reported [37][38][39] . These assays were developed to provide (Table 1).…”
We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.
“…However, the use of a pseudovirus also raises numerous challenges. Factors such as the folding, cleavage, density and geometry of spike proteins on the virion can affect both the mechanics of cell entry and the ability of antibodies to bind to (pseudo)virions and neutralize infectivity 15 , 16 and may differ from those of the native virus 17 – 20 . Fig.…”
Section: Measuring Antiviral Activity In Vitromentioning
confidence: 99%
“…Numerous assays involve measuring the ability of antibodies to inhibit the replication of virus over several days. Both replicating VSV/SARS-CoV-2 chimeric viruses 1 , 15 and native SARS-CoV-2 (refs 1 , 21 ) have been used to infect susceptible cell lines, with subsequent measurement of the level of infection after several days of incubation by quantifying reporter protein expression, viral antigen in infected cells or free virus in the supernatant (see Table 1 ). These assays can then be used to measure antibody neutralization by pre-incubation of different concentrations of antibody with the viral inoculum and measuring the relationship between antibody concentration and inhibition of infection.…”
Section: Measuring Antiviral Activity In Vitromentioning
The rapid scale-up of research on coronavirus disease 2019 (COVID-19) has spawned a large number of potential vaccines and immunotherapies, accompanied by a commensurately large number of in vitro assays and in vivo models to measure their effectiveness. These assays broadly have the same end-goal — to predict the clinical efficacy of prophylactic and therapeutic interventions in humans. However, the apparent potency of different interventions can vary considerably between assays and animal models, leading to very different predictions of clinical efficacy. Complete harmonization of experimental methods may be intractable at the current pace of research. However, here we analyse a selection of existing assays for measuring antibody-mediated virus neutralization and animal models of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a framework for comparing results between studies and reconciling observed differences in the effects of interventions. Finally, we propose how we might optimize these assays for better comparison of results from in vitro and animal studies to accelerate progress.
“…Also, the ability of lentivirus to elicit immune response in infected cells is limited. Further studies of SARS-CoV-2 virus cell entry and its inhibition with a replication competent surrogate coronavirus [49,50] are warranted.…”
COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. Chloroquine or hydroxychloroquine increase cleaved active SP-domain of TMPRSS2, and this is potentiated by MEKi. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1alpha, IL-6 and MCP-1 are suppressed by MEKi alone or in combination with remdesivir. MEKi enhance NK cell (but not T-cell) killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. We generated a pseudotyped SARS-CoV-2 virus with a lentiviral core but with the SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope and used VSV-G lentivirus as a negative control. Our results show infection of human bronchial epithelial cells or lung cancer cells and that MEKi suppress infectivity of the SARS-CoV-2-S pseudovirus following infection. We show a drug class-effect with MEKi to promote immune responses involving NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells in a model system. MEKi may attenuate coronavirus infection to allow immune responses and antiviral agents to control COVID-19 disease progression and severity.
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