Effective prophylaxis and antiviral therapies are urgently needed in the event of reemergence of the highly contagious and often fatal severe acute respiratory syndrome (SARS) coronavirus (SARSCoV) infection. We have identified eight recombinant human single-chain variable region fragments (scFvs) against the S1 domain of spike (S) protein of the SARS-CoV from two nonimmune human antibody libraries. One scFv 80R efficiently neutralized SARS-CoV and inhibited syncytia formation between cells expressing the S protein and those expressing the SARS-CoV receptor angiotensin-converting enzyme 2 (ACE2). Mapping of the 80R epitope showed it is located within the N-terminal 261-672 amino acids of S protein and is not glycosylation-dependent. 80R scFv competed with soluble ACE2 for association with the S1 domain and bound S1 with high affinity (equilibrium dissociation constant, K d ‫؍‬ 32.3 nM). A human IgG1 form of 80R bound S1 with a 20-fold higher affinity of 1.59 nM comparable to that of ACE2 (K d ‫؍‬ 1.70 nM), and neutralized virus 20-fold more efficiently than the 80R scFv. These data suggest that the 80R human monoclonal antibody may be a useful viral entry inhibitor for the emergency prophylaxis and treatment of SARS, and that the ACE2-binding site of S1 could be an attractive target for subunit vaccine and drug development.T he severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), a newly emergent member in the family Coronaviridae, causes SARS for which there are no vaccines or effective therapies currently available (1-4). It has been reported that high titers of protecting IgG antibody to SARS-CoV are present in convalescent serum, and SARS patients show clinical improvement if they are given serum from previously infected patients (5, 6). These observations suggest that passive immunization with human monoclonal antibodies could be developed for the treatment of SARS (7). The spike (S) proteins of coronaviruses are large type-I transmembrane glycoproteins that are responsible for receptor binding and membrane fusion. Two functional domains at the amino (S1) and carboxy (S2) termini of the S protein are conserved among the coronaviruses. The S1 and S2 domain of SARS-CoV S protein can be identified by sequence alignment with other coronavirus S proteins, especially with the more conserved S2 domain (8-10). The S protein is also the major antigenic determinant for coronaviruses (9,(11)(12)(13)(14). It has recently been demonstrated that the binding of the S1 domain to its receptor angiotensinconverting enzyme 2 (ACE2) on host cells is responsible for SARS-CoV entry into cells (15). Therefore, we targeted the S1 protein for generation of neutralizing human monoclonal antibodies. Here we report the identification, production, and characterization of a neutralizing human monoclonal antibody 80R against SARS-CoV that blocks the binding of S1 to ACE2. Materials and MethodsExpression and Purification of SARS-CoV S1 and Truncated S1. Plasmids encoding SARS-CoV S protein residues 12-672, 12-327, o...

In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding