Influenza virus remains a constant public health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Monoclonal antibody (mAb)-based immunotherapy is a promising strategy for disease control. Here we use a human Ab phage display library and H5 hemagglutinin (HA) ectodomain to select ten neutralizing mAbs (nAbs) with a remarkably broad range among Group 1 influenza viruses, including the H5N1 “bird flu” and the H1N1 “Spanish flu” strains. Notably, nine of the Abs utilize the same germline gene, VH1-69. The crystal structure of one mAb bound to H5N1 HA reveals that only the heavy chain inserts into a highly conserved pocket in the HA stem, inhibiting the conformational changes required for membrane fusion. Our studies indicate that nAbs targeting this pocket could provide broad protection against both seasonal and pandemic influenza A infections.

Human angiotensin‐converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS‐CoV). Here we identify the SARS‐CoV spike (S)‐protein‐binding site on ACE2. We also compare S proteins of SARS‐CoV isolated during the 2002–2003 SARS outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, a possible source of SARS‐CoV found in humans. All three S proteins bound to and utilized palm‐civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S‐protein‐binding site of human ACE2 to those of civet ACE2, or by altering S‐protein residues 479 and 487 to residues conserved during the 2002–2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS‐CoV to human cells, and provide insight into the severity of the 2002–2003 SARS epidemic.

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...