REMDESIVIR RETAINS POTENT ACTIVITY AGAINST SARS-CoV-2 VARIANTS OF CONCERN

ABSTRACT

Background: Recent SARS-CoV-2 variants of concern (VOCs) have shown a progressive loss of sensitivity to monoclonal antibody therapeutics. Remdesivir (RDV) is a nucleotide analog prodrug that targets the viral RNA-dependent RNA polymerase (RdRp) Nsp12 and is approved to treat COVID-19 in hospitalized and non-hospitalized patients. Nsp12 is highly conserved across VOCs to date and RDV antiviral activity against previous VOCs (Alpha to Omicron BA.1) has been maintained. Here, we conduct a structural analysis of Nsp12 substitutions observed in recent Omicron subvariants (BA.2, BA.2.12.1, BA.4, BA.5 and BA.2.75) and assess RDV antiviral activity against clinical isolates and sitedirected mutants (SDMs) in a replicon system. Method(s) The prevalence of Nsp12 substitutions in Omicron subvariants was evaluated by analysis of sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) EpiCoV database. Structural analysis of identified substitutions was conducted on a prior cryo-electron microscopy-based model of the replication-transcription complex. Antiviral activity against subvariant clinical isolates was assessed by nucleoprotein ELISA in A549-hACE2-TMPRSS2 cells and by SDMs in the replicon system. Result(s) Genomic analysis of >1.4 million Omicron subvariant sequences revealed unique substitutions in Nsp12 compared to the ancestral WA1 strain. Besides P323L, present in all subvariants, G671S was observed in 95.9% of BA.2.75 sequences, F694Y was observed in <=1.9% of BA.4, BA.5 and BA.2.75 sequences, and Y521C was observed in 1.7% of BA.5 sequences. As anticipated, structural analysis of these substitutions showed no direct interaction with the incoming RDV nucleotide triphosphate or the viral RNA. Phenotyping of clinical isolates of Omicron subvariants BA.2, BA.2.12.1, BA.4, BA.5, and BA.2.75 consistently resulted in mean RDV EC50 values of 24.5 nM (BA.2) to 106.0 nM (BA.5). This represented 0.15-to 0.66-fold changes compared to WA1, indicating no loss of in vitro RDV antiviral activity against these VOCs. P323L, G671S, and F694Y were shown previously to have no impact on RDV antiviral activity. Similarly, the individual substitution Y521C showed no change in RDV susceptibility in the SARS-CoV-2 replicon system. Conclusion(s) RDV retained potent in vitro antiviral activity against all tested Omicron VOCs with potencies comparable to the WA1 isolate. These data support the continued use of RDV in patients infected with Omicron subvariants.