Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

Abstract

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein.One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.Severe acute respiratory syndrome (SARS) was first identified in 2002 as a newly emerging disease in Guangdong Province, China. The disease, associated with unusual atypical pneumonia, spread in 2003 to over 30 countries worldwide with more than 8,000 reported cases and an estimated 55% mortality among the elderly (9). A virus was isolated from tissues of SARS patients (10, 21, 23, 32) and a SARS-associated coronavirus (SARS-CoV), a new member in the family of Coronaviridae, was identified as the causative agent fulfilling Koch's postulates (12).The clinical course of SARS is highly variable (31) after a relatively short 6-to 10-day incubation period (9). In ca. 20% of the patients, SARS-CoV infection progresses to a stage of respiratory failure requiring ventilation support. Overall, 10% of the patients, ca. 6.8% of patients younger and 55% of patients older than 60 years of age (9), die as a consequence of immunopathological lung damage caused by a hyperactive antiviral immune response (29).Antibodies to SARS-CoV become detectable in patient's serum between days 10 and 15 and correlate with a decline in viral loads. More than 93% of the patients were reported to have seroconverted by day 28 (31). The pattern of SARS-CoV replication and development of a neutralizing immune response observed in experimentally infected mice largely resembles...

SARS Vaccine Development

“…Using spleen cells from mice immunized with RBD, we have generated a panel of 25 monoclonal antibodies (MAbs) that recognize different conformational epitopes on RBD and possess potent neutralizing activity (38). Our result is in agreement with the report by van den Brink et al (39), who identified 3 human neutralizing anti-S MAbs from antibody phage display libraries by using inactivated SARS-CoV as the target. These researchers also found that all of these MAbs specifically bound to RBD and blocked interaction between RBD and ACE2.…”

“…When the early and late SARS-CoV strains are compared, only 3 to 5 aa residues are variable among the 193 residues in RBD and most of the isolates vary by only 1 residue (4). van den Brink et al (39) showed that 1 human MAb (CR3014) specific for RBD of SARS-CoV strain FM1 can effectively bind to most RBDs of the early and late SARS-CoV strains. These data suggest that antibodies directed against RBD of a SARS-CoV isolate may neutralize infection by a broad spectrum of SARS-CoV strains.…”

Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies

“…The neutralizing activity of this antibody in our cell fusion assay was about 2-fold lower compared with that of m396 (IC 50 ϭ 1.1 vs. 0.6 g/ml) for the Tor2 isolate. Another representative antibody, CR3014, protected from the cytopathic effects of two SARS-CoV isolates, FM1 and HKU39849; the concentration for protection from HKU39849 was Ϸ7 g/ml (21,26). The scFv B1 neutralized with an IC 50 of Ϸ4 g/ml in a pseudovirus assay (22).…”

The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries

“…Titration of IgG 1 binding using FCM was performed using the RV gp-transfected PER.C6 cells in a procedure as described before [36]. Titration in ELISA was performed using the ELISA described above with a horseradish peroxidase (HRP)-coupled mouse anti-human secondary antibody (Jackson ImmunoResearch).…”