Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants

Abstract

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC₅₀) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC₅₀ values ranging from 0.13- to 2.3-fold of the observed EC₅₀ values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.

Keywords: COVID-19; GS-441524; Nsp12; SARS-CoV-2 variants; antiviral agents; remdesivir.

FIG 1

Representative dose-response curves of remdesivir and GS-441524 against SARS-CoV-2 VOC. Shown are dose-response curves of RDV (A and B) and GS-441524 (C) activity against the WA1 reference isolate and SARS-CoV-2 VOCs in A549-ACE2-TMPRSS2 cells by plaque reduction assay (PRA) (A) or ELISA (B and C). In the PRA, infected cell supernatants were harvested at 48 hpi (solid lines) or 72 hpi (dashed lines) and analyzed by plaque assay on Vero-TMPRSS2 cells. For ELISA, infected cells were fixed at ~48 hpi (solid lines) or ~72 hpi (dashed lines) and processed. The data shown are means and standard deviations from representative experiments that were performed in biological quadruplicates (PRA) or triplicates (ELISA) at each compound concentration. Average calculated EC₅₀ values and fold change from WA1 reference can be found in Table 1.

FIG 2

Structural model of Nsp12 highlighting key amino acid substitutions in relation to the active site. Preincorporated remdesivir triphosphate (RDV-TP) was modeled into the cryo-EM structure of the polymerase complex (PDB no. 6XEZ) (37). Nsp12 is represented in green, with Nsp8 in yellow, and Nsp7 in white. The prevalent amino acid substitution P323L, seen in all variants, was measured to be 28.6 Å from RDV-TP (P323 Cα-RDV-TP C1′), whereas G671S, seen in the Delta variant, is at 24.9 Å. Of all the amino acid substitutions reported here, F694Y, seen at low frequency in Delta and Omicron, comes closest to the active site, at 12.2 Å. A computational analysis suggests that the substitutions have no meaningful impact on RDV-TP binding affinity.

FIG 3

Recombinant Omicron viruses retain susceptibility to RDV and GS-441524. Shown are ELISA dose-response curves of RDV (solid lines and solid symbols) and GS-441524 (dashed lines and open symbols) activity against recombinant Omicron viruses with (dark purple) or without (light purple) the F694Y substitution compared with WA1 (black), and Omicron (gray) clinical isolates run in parallel. The data shown are means and standard deviations from a representative 72-hpi nucleoprotein ELISA that was performed with biological triplicates at each compound concentration. Average calculated EC₅₀ values are in Table 3.

FIG 4

Prevalent Nsp12 substitutions in Delta and Omicron retain susceptibility to RDV and GS-441524. Shown are dose-response curves of RDV (solid lines and solid symbols) and GS-441524 (dashed lines and open symbols) activity against recombinant viruses with/without prevalent Nsp12 substitutions containing a Nano luciferase (Nluc) (A) or firefly luciferase (Fluc) (B) transgene. The data shown are means and standard deviations from a representative experiment that was performed in biological duplicates at each compound concentration. Average calculated EC₅₀ values and fold change from recombinant WA1 references are in Table 4.