Identification of antibody-resistant SARS-CoV-2 mutants via N4-Hydroxycytidine mutagenesis
Priya Kumar, Xiaoxiao Zhang, Rahul Shaha, Maik Kschischo, Matthias Dobbelstein
Antiviral Research, doi:10.1016/j.antiviral.2024.106006
Monoclonal antibodies targeting the Spike protein of SARS-CoV-2 are effective against COVID-19 and might mitigate future pandemics. However, their efficacy is challenged by the emergence of antibody-resistant virus variants. We developed a method to efficiently identify such resistant mutants based on selection from mutagenized virus pools. By inducing mutations with the active compound of Molnupiravir, N4-hydroxycytidine (NHC), and subsequently passaging the virus in the presence of antibodies, we identified specific Spike mutations linked to resistance. Validation of these mutations was conducted using pseudotypes and immunofluorescence analysis. From a Wuhan-like strain of SARS-CoV-2, we identified the following mutations conferring strong resistance towards the corresponding antibodies: Bamlanivimab -E484K, F490S and S494P; Sotrovimab -E340K; Cilgavimab -K444R/E and N450D. From the Omicron B.1.1.529 variant, the strongly selected mutations were: Bebtelovimab -V445A; Sotrovimab -E340K and K356M; Cilgavimab -K444R, V445A and N450D. We also identified escape mutations in the Wuhan-like Spike for the broadly neutralizing antibodies S2K146combined G485S and Q493R -and S2H97 -D428G, K462E and S514F. Structural analysis revealed that the selected mutations occurred at antibody-binding residues within the receptor-binding domains of the Spike protein. Most of the selected mutants largely maintained ACE2 binding and infectivity. Notably, many of the identified resistance-conferring mutations are prevalent in real-world SARS-CoV-2 variants, but some of them (G485S, D428G, and K462E) have not yet been observed in circulating strains. Our approach offers a strategy for predicting the therapeutic efficacy of antibodies against emerging virus variants.
Declaration of competing interest The other authors declare no conflict of interest.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.antiviral.2024.106006.
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