TMPRSS2-mediated coronavirus spike activation and inhibition

The coronavirus spike (S) glycoprotein mediates infection by fusing viral and host membranes through large-scale conformational changes triggered by receptor binding and proteolysis. S is a metastable prefusion trimer anchored in the viral membrane with its receptor-binding domains dynamically sampling conformations that balance host receptor engagement and immune evasion as it is the main target of neutralizing antibodies. Given the key role of the S protein in viral entry, alteration of receptor recognition modulates host and tissue tropism and can lead to spillover. The protease TMPRSS2 facilitates coronavirus infections, yet its mechanism of viral glycoprotein recognition remains unclear. We show that, following ACE2 engagement of the SARS-CoV-2 spike (S) inducing the early fusion intermediate conformation (E-FIC), TMPRSS2 cleaves the R815 S2′ site and promotes fusogenic conformational changes leading to viral entry. We unveil TMPRSS2 recognition of S2′, identify key residues modulating binding specificity and demonstrate that S2′ site-directed broadly neutralizing antibodies target E-FIC and inhibit viral entry by blocking TMPRSS2 access. We computationally designed stabilized E-FIC as a vaccine candidate, overcoming the transient nature of this state. We describe a TMPRSS2-directed monoclonal antibody inhibiting several coronaviruses, including SARS-CoV-2 variants and protecting mice against SARS-CoV-2 challenge. These results outline the mechanistic role of TMPRSS2 and S2′ site-directed antibodies in coronavirus entry and pave the way for developing broad spectrum countermeasures.