Identification of Antibody-Resistant SARS-CoV-2 Mutants via N4-Hydroxycytidine Mutagenesis

Monoclonal antibodies (mAbs) to the virus Spike protein have been prominent for treating SARS-CoV-2 infections, especially in immunocompromised patients. However, since the onset of the pandemic, numerous variants have emerged, with immune-evasive mutations on their spike proteins. Identification of such spike mutations could be key in understanding how the virus confers resistance against protective Abs, thereby predicting therapeutic success. In this study, we made use of the antiviral drug Molnupiravir to enhance virus mutagenesis and obtain a pool of virus mutants, as we described (Zibat et al., iScience 2023). The mutants were then passaged in the presence of different therapeutic and broad spectrum mAbs. This quickly led to the accumulation of resistant viruses. Deep sequencing revealed characteristic mutations within the Spike gene.