Numerous genetic mutations have emerged in the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since its detection in Wuhan, China, resulting in the emergence of new variants- Omicron being the most recent variant. These pathogenic variants have been termed variants of concern (VOC) or variants of interest (VOI). Changes in the viral genome have enabled increased viral replication and have reduced its susceptibility to the host immune system, antiviral drugs, and vaccine-generated antibodies.
Researchers have found that these variants result from changes in the gene encoding the spike protein—which mediates the entry of the virus into the host cell—and also due to amino acid substitutions in Nsp12 – the RNA- dependent RNA polymerase (RdRP) and Nsp5 3CL main protease (Mpro).
Remdesivir (RDV) was the first antiviral drug approved for treating coronavirus disease 2019 (COVID-19). RDV is a prodrug of its parent nucleoside GS-441524. Nsp12 RdRp and Nsp5 3 CL Mpro are the two targets of the antivirals approved for treating COVID-19 infection. RDV acts by incorporating its triphosphate metabolite (RDV-TP) into the RNA of the virus, which interferes with the Nsp12 at multiple locations, thereby compromising its further synthesis.
A new study published inbioRxiv*preprint serverdemonstrated thein vitro potencies of RDV and GS-441524 through plaque reduction assay (PRA), and nucleoprotein enzyme-linked immunosorbent assay (ELISA) against SARS-CoV-2 and other VOC/VOIs isolates with specific substitutions in Nsp12.
For this study, RDV and GS-441524 were synthesized, and viral culture was carried out. Plaque formation assay (PFA), PRA, nucleoprotein ELISA, SARS-CoV-2 sequence analysis, protein structure modeling, site-directed mutagenesis, recombinant virus rescue, construction of a recombinant Omicron SARS-CoV-2, antiviral activity assessment from recombinant luciferase containing viruses, and EC50determinations were also done.
The antiviral activity of RDV was assessed initially using PRA. Infected A549-ACE2-TMPRSS2 cultures were used from which supernatants were harvested at 48 hours post-infection at a multiplicity of infection (MOI) of 0.1. The result indicated potent antiviral activity of RDV against all variants of SARS-CoV-2. The Delta variant was nearly three times more susceptible to RDV than the initial WA1 isolate.
Nucleoprotein ELISA was used to assess the antiviral effect directly in the infected cultures. The Omicron variant was found to be significantly more susceptible to both RDV and GS-441524 than the WA1 strain – early ancestral A lineage isolates detected in Wuhan.
Overall, 5,842,948 SARS-CoV-2 variant sequences were analyzed to assess the genetic variants in the 11 VOC/VOIs. It was found that the Delta variant comprised the highest proportion of analyzed sequences followed by Alpha, Omicron, and the other eight variants.
Genetic variations in the spike in comparison to Nsp12 were also analyzed. Overall, substitutions of one to six amino acids were observed among the variants with 7-45 substitutions over the 1,274 amino acid positions in the spike and a frequency of ³ 1% sequences over the 932 amino acid positions in Nsp12. A frequency of > 99% was estimated with the most prevalent Nsp12 substitution relative to the sequence P323L, which defined Nsp12 substitution for the analyzed 11 variants.
In 97.8% of the Delta isolates, another lineage-defining amino acid change in Nsp12, G671S was found. No other substitution was noted in any other variant. Further investigation of six substitutions in the sequences of Omicron variant revealed a 99.5% substitution in P323L, 2.0% substitution in the F694Y, while the remaining four substitutions had £ 1% frequency.
In the initial stages of the Omicron variant emergence (December 13, 2021), F694Y was highly prevalent, which rapidly declined in frequency. As of January 18, 2022, the frequency of F694Y substitution was only 2% of the deposited sequences. Nsp12 substitution susceptibility to RDV was found to be 0.0002% for F480L, 0.0004% for V557L and .002% for E802D.
Furthermore, at S861 – the residue involved in RDV induced delayed chain termination; only 0.0008% of the sequences had any alteration.
Using a model based on the structure of RdRp complex (Nsp12/(Nsp8)2/Nsp7/(Nsp13)2), the potential impact of each Nsp12 substitution was assessed on the affinity of RDV-TP substitution for RdRp active site in all the variants. From the pre-incorporated RDV-TP, the two most common substitutions, P323L (seen in all variants) and G671S (present in the Delta variant), were found to be 28.6 angstrom and 24.9 angstrom, respectively.
Meanwhile, F694Y was near the RdRp active site (12.2 angstrom). However, on evaluating its impact on RDV-TP binding affinity, no significant difference was found. Most low-frequency substitutions among variants of SARS-CoV-2 occur away from the polymerase active site, on the Nsp12 surface, suggesting a minimal impact on the efficacy of RDV and GS-441524.
The RDV and GS-441524 activity against recombinant Omicron (rOmicron) variant were assessed with and without the Nsp12 F694Y substitution. Both remdesivir and its parent nucleoside were potent against the two recombinant Omicron viruses and an Omicron clinical isolate. All three viruses showed increased susceptibility to both drugs compared to WA1 isolate by ELISA.
Further, the potency of both drugs against other Nsp12 substitutions alone or in combination in any specific variant was found to be at high frequency (>15%). The results concluded that modifications of the Nsp12 amino acid with a sequence encoding G-671S alone could not rescue infectious viruses. Recombinant viruses containing P323L or F694Y alone or P323L/F694Y double substitution were susceptible to RDV. GS-441524 was found to be potent against recombinant SARS-CoV-2with Nsp12 P323L, P323L/G671S, and P323L/F694Y substitutions. Overall, the data suggested RDV and GS-441524 are potent against viruses with prevalent Nsp12 substitutions.
This study highlights the potency of RDV and GS-441524 against the variants of SARS-CoV-2 through numerous mechanisms, and hence, supports its use in the approved patients.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.