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.

FIG. 1.

Selective binding of isolated scFv phage antibodies to SARS-CoV by ELISA. (A) Phage produced from individual colonies were analyzed for binding to SARS-CoV or FBS coated on microtiter wells. (B) Binding of IgGs constructed from the isolated scFv to SARS-CoV.

FIG. 2.

Deduced protein sequences of VH and VL domains of clones that bind to SARS-CoV. FR, framework region; CDR, complementarity-determining region. Dots indicate sequence identity to the consensus sequence.

FIG. 3.

Binding of the IgGs to SARS-CoV-infected Vero cells. Immunofluorescent staining with IgG CR3009 (A), CR3014 (B), CR3018 (C), or negative control IgG (D) is shown.

FIG. 4.

Binding of human IgGs to N protein analyzed by ELISA. (A) Binding of human IgGs to N protein or an irrelevant negative control protein captured via an anti-myc antibody. (B) Titration of IgGs CR3009, CR3018, and negative control IgG to the N protein. (C) Competition ELISA on immobilized SARS-CoV for binding to N protein. The binding of biotinylated IgG CR3009 was analyzed in the presence of increasing amounts of competitor IgG CR3009, CR3018, or CR3014. The binding of biotinylated IgG CR3018 was analyzed in the presence of competitor IgG CR3009, CR3018, or CR3014. The binding is expressed as the percentage of binding without competitor. (D) Pepscan analysis of N-protein-derived peptides with IgG CR3018. On the y axis, the 18 amino-terminal peptides of N protein are depicted. The binding to linear or looped peptides is indicated as the absorbance at 405 nm (×1,000) on the x axis.

FIG. 5.

Visualization of N protein by ultrathin-section immuno-EM. Gold immunolabeling of N protein in SARS-CoV-infected Vero cells with CR3009 (A), CR3018 (B), or negative control MAb (C) was carried out, followed by incubation with 5-nm-colloidal-gold-conjugated secondary antibody.

FIG. 6.

Analysis of the binding of human IgGs to S protein. (A) Binding of IgGs to S-protein-transfected 293T cells with fluorescence-activated cell sorting indicated as the mean fluorescence intensity or to recombinant S565 or S318-510 fragment as determined by ELISA. (B) Titration of IgGs CR3006, CR3013, CR3014, and negative control IgG (Control) in an S565 fragment-coated ELISA. (C) Competition ELISA on immobilized S565. The binding of biotinylated IgG CR3006 (solid symbols) was analyzed in the presence of increasing amounts of competitor IgG CR3006, CR3013, or CR3014. The binding of biotinylated IgG CR3013 (dotted lines) was analyzed in the presence of competitor IgG CR3006, CR3013, or CR3014, and the binding of biotinylated IgG CR3014 (open symbols) was analyzed in the presence of competitor IgG CR3006, CR3013, or CR3014. Binding is expressed as the percentage of binding without competitor.

FIG. 7.

Binding of human IgGs to recombinant S318-510 fragment with naturally occurring mutations. (A) Binding of CR3006, CR3013, and CR3014 to recombinant S318-510 fragments and variants thereof by ELISA. (B) Binding of CR3014 and anti-HIS6 MAb to recombinant S318-510 fragments, which was normalized for binding to FM1 S318-510.

FIG. 8.

Inhibition of the interaction of S565 with Vero cells by human IgG measured by fluorescence-activated cell sorting. Vero cells were incubated with S-protein-derived fragment S565 in the absence (thin line) or presence (solid line) of IgG CR3014 (A), CR3018 (B), or CR3006 (C). The dotted line represents the negative control (irrelevant myc-tagged protein).

FIG. 9.

SARS-CoV neutralizing activity of scFv and IgGs. Neutralization is shown as percentage. Protection against 100 TCID₅₀ SARS-CoV in two of three culture wells by scFv CR3013 (⋄) and CR3014 (♦) is indicated. No protection by scFv CR3006 (✽) was observed. The neutralization value shown for each IgG is the percentage of culture wells protected from infection with SARS-CoV. The experiments were performed twice in triplicate. Results for neutralization by serially diluted IgG CR3014 of 10 (•), 30 (▴), or 100 (▪) TCID₅₀ of SARS-CoV and 100 and 10 TCID₅₀ of SARS-CoV by CR3013 (□) and control IgG (×), respectively, are shown.