Importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on MERS-CoV Spike to avoid neutralization escape.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or healthcare-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell-cloning strategy, we have identified and characterized multiple neutralizing mAbs specifically binding to the receptor binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific mAbs tended to have greater neutralizing potency than non-RBD S1-specific mAbs. Six RBD-specific and five S1-specific mAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined co-crystal structures for two mAbs targeting RBD from different angles and show they can only bind RBD in the "out" position. We then showed that selected RBD-specific, non-RBD S1, and S2-specific mAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD mAbs delayed the emergence of escape mutations in a cell-based virus-escape assay. These studies identify mAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization, and for developing more effective interventions to prevent or treat MERS-CoV infections. MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally-defined probes for the MERS-CoV Spike (S) glycoprotein, the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized non-human primates (NHPs). mAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining mAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed mAbs. These data suggest that antibody responses to multiple domains on CoV Spike may improve immunity and will guide future vaccine and therapeutic development efforts.