Characterization of human neutralizing antibodies against severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerged pathogen that causes respiratory distress in infected individuals with an overall mortality rate of approximately 10 percent. The spike (S) protein of SARS-CoV facilitates entry into susceptible cells via the cellular receptor ACE2. The S protein is divided into two functional domains; S1 mediates receptor binding and S2 mediates fusion of viral and host cell membranes. SARS-CoV S protein is the main antigenic target of the virus and neutralizing antibodies in both animal models and human infection are targeted to S protein. Neutralizing antibodies prevent viral replication both in vitro and in vivo, and clearly indicate that the humoral immune response plays a major role in protection against SARS-CoV replication. Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be effective in SARS-CoV infected individuals. Utilizing the human immunoglobulin transgenic mouse, XenoMouseRTM, we produced a large panel of neutralizing mAbs against SARS-CoV S protein. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the receptor binding domain (RBD), as 318-510 and inhibited by preventing receptor association. Two S1 specific mAbs reacted with a domain upstream of the RBD between as 12 to 261. The neutralization mechanism for each of these mAbs is different, one inhibits receptor binding, and one inhibits a post-binding event. Immunoglobulin gene sequence analyses and competitive inhibition studies of the mAbs suggested 7 different binding specificities. The mAbs demonstrated variability in the generation of escape mutants that can be neutralized by a cocktail of mAbs. Furthermore, a single amino acid change demonstrated that changes within the S protein may alter mAb binding, exemplifying the requirement to carefully examine mAbs to be used as a therapeutic cocktail.