P681 mutations within the polybasic motif of spike dictate fusogenicity and syncytia formation of SARS CoV-2 variants

The rapid spread and dominance of the Omicron SARS-CoV-2 over its Delta variant has posed severe global challenges. While extensive research on the role of the Receptor Binding Domain on viral infectivity and vaccine sensitivity has been documented, the role of the spike₆₈₁PRRAR/SV₆₈₇polybasic motif is less clear. Here we monitored infectivity and vaccine sensitivity of Omicron SARS-CoV-2 pseudovirus against sera samples that were drawn four months post administration of the third dose of BNT162b2 mRNA vaccine. Our findings show that relative to Wuhan-Hu and Delta SARS-CoV-2, Omicron displayed enhanced infectivity and a sharp decline in its sensitivity to vaccine-induced neutralizing antibodies. Furthermore, while the spike proteins form Wuhan-Hu (P681), Omicron (H681) and BA.2 (H681) pseudoviruses modestly promoted cell fusion and syncytia formation, Delta spike (P681R) displayed enhanced fusogenic activity and syncytia formation capability. Live-viruses plaque formation assays confirmed these findings and demonstrated that relatively to the Wuhan-Hu and Omicron SARS-CoV-2, Delta formed more plaques that were smaller in size. Introducing a single P681R point mutation within the Wuhan-Hu spike, or H681R within Omicron spike, restored fusion potential to similar levels observed for Delta spike. Conversely, a R681P point mutation within Delta spike efficiency abolished fusion potential. We conclude that over time, the efficiency of the third dose of the Pfizer vaccine against SARS CoV-2 is waned, and cannot neutralize Omicron. We further verify that the P681 position of the viral spike dictates fusogenicity and syncytia formation.