Determination of resilience of a panel of broadly neutralizing mAbs to emerging variants of SARS-CoV-2 generated using reverse genetics.

SARS-CoV-2 continues to evolve, and its emerging variants might escape the immune responses generated by existing vaccines and therapeutic mAbs. Accordingly, rapid analysis of their possible neutralization phenotype is essential and can be facilitated by reverse genetics (RGs) systems to regenerate viruses with variant-specific substitutions. Here, we efficiently generate a panel of recent variants of SARS-CoV-2 (Omicron XBB.1.16, EG.5.1, BA.2.86, and JN.1) using a substantially optimized circular polymerase extension reaction (CPER) RGs system. Neutralization potency was analyzed for mAbs targeting different regions of spike protein. mAbs P4-J15, C68.61, S2X259, and IY-2A IgG were able to neutralize all recent viruses. However, S309, which was previously used to treat infection and targets the outer face of RBD, showed ∼75-fold reduction in potency versus JN.1. Moreover, C68.59, which targets the SD1 region of the CTD, was unable to neutralize either BA.2.86 or JN1, which share the E554K substitution in SD1. CPER RGs system and microneutralization assays can be adopted as effective tools to evaluate the efficacy of therapeutic mAbs against emerging variants in a time-responsive manner.