Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse

Abstract

Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human immunoglobulin transgenic mouse, XenoMouse, we produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examined for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID(50) SARS-CoV (Urbani). Additionally, 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318-510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Immunoglobulin gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants.

Fig. 1

Reactivity of HmAbs produced from hybridomas generated from immunized XenoMouse® against S1-Ig fragments by ELISA. All S-V5-HIS reactive Abs were tested against S1-Ig (12–672) coated recombinant protein. The 200 Abs that reacted with S1-Ig (12–672) were then tested for reactivity against three S1-Ig fragments (12–510, 261–510 and 318–510).

Fig. 2

Expression of overlapping fragments of the S1 domain of SARS-CoV S protein. (A) Four plasmid constructs encoding different fragments of the S1 protein (12–672, 12–510, 261–672, 318–510) were transformed into MC 1061/P3 cells and insert size confirmed by digestion with NheI and BamH1 and analyzed on a 1% agarose gel. (B) Recombinant protein expression in transiently transfected 293T cells was confirmed by Coomassie Blue staining of a 4–20% SDS/PAGE gel.

Fig. 3

Neutralizing mAbs were purified and examined for S1-Ig fragment reactivity in an ELISA. Recombinant proteins were coated on ELISA plates and increasing dilutions of mAb tested for reactivity to the relevant S1 recombinant protein. (A) Group 1A purified mAbs reactivity to 318–510 recombinant protein. (B) Group 1B purified mAbs reactivity to 318–510 recombinant protein. (C) Group 1D purified mAb reactivity to 318–510 recombinant protein. (D) Goup 2B purified mAbs reactivity to 12–510 recombinant protein.

Fig. 4

Alignment of CDR sequences of neutralizing mAbs. Immunoglobulin genes of neutralizing antibodies were sequenced. Alignment of the amino acid sequences of the heavy chain variable region (A) and light chain variable region (B) of all mAbs are depicted and arranged by common gene segment usage. Additions in mAb sequences not contained in germ line sequence are annotated (#) in germ line sequence. N/A specific gene segment could not be identified.