Susceptibility of broad reactivity nanobodies to resistance mutations in the S2 domain of SARS-CoV-2 predicted by yeast display deep mutational scanning

Introduction: The rapid evolution of SARS-CoV-2 has led to the erosion of vaccine induced serum neutralization and monoclonal antibody efficacy. As such, interest is inevitably moving towards more conserved regions of the SARS-CoV-2 spike protein like the S2 domain. Resistance mutations continue to be a major obstacle for the development of antivirals and vaccines which target the RBD but what extent these will be a problem for S2 binding antibodies is not known. Methods: We have developed a yeast display deep scanning mutagenesis platform which allows an unbiased prospective assessment of millions of single and double mutations for their effects on antibody binding to the S2 domain. Results: We have compared the mutational resistance of a panel of five nanobodies mapped to four distinct non-competing epitopes within the conserved fusion peptide, stem helix and heptad repeat 2 elements of the S2 domain. Yeast display deep mutational scanning predicted reduced binding of C303, G223, G225, and G142 to naturally occurring resistance mutations which were experimentally confirmed on SARS-CoV-2 variants. Discussion: Our study shows that resistance mutations in conserved elements of the S2 domain may still pose a challenge to the development of monoclonal antibodies and subunit vaccines.