Molecular characterization of a panel of murine monoclonal antibodies specific for the SARS-coronavirus

Abstract

The availability of monoclonal antibodies (mAbs) specific for the SARS-coronavirus (SARS-CoV) is important for the development of both diagnostic tools and treatment of infection. A molecular characterization of nine monoclonal antibodies raised in immune mice, using highly purified, inactivated SARS-CoV as the inoculating antigen, is presented in this report. These antibodies are specific for numerous viral protein targets, and six of them are able to effectively neutralize SARS-CoV in vitro, including one with a neutralizing titre of 0.075 nM. A phylogenetic analysis of the heavy and light chain sequences reveals that the mAbs share considerable homology. The majority of the heavy chains belong to a single Ig germline V-gene family, while considerably more sequence variation is evident in the light chain sequences. These analyses demonstrate that neutralization ability can be correlated with specific murine V(H)-gene alleles. For instance, one evident trend is high sequence conservation in the V(H) chains of the neutralizing mAbs, particularly in CDR-1 and CDR-2. The results suggest that optimization of murine mAbs for neutralization of SARS-CoV infection will likely be possible, and will aid in the development of diagnostic tools and passive treatments for SARS-CoV infection.

Fig. 1

Alignments of the nucleotide and amino acid sequences of V_H regions of the neutralizing mAbs belonging to the J558 V-gene family. (A) Nucleotide alignment of the V_H regions of the mAbs most closely related to the J558.50 V-gene. The consensus sequence used is that of J558.50 (nucleotide sequence GenBank accession no. AF303881). A dot in the individual sequences denotes nucleotides that are the same as the consensus. A dash in the individual sequences denotes a deletion. Neutralizing clones are shown in bold text. (B) Amino acid alignment of the V_H regions of the mAbs most closely related to the J558.50 Ig V-gene. The consensus sequence used is that of J558.50 (amino acid sequence GenBank accession no. AAG39162). A dot in the individual sequences denotes amino acids that are the same as the consensus. A dash in the individual sequences denotes a deletion. The framework and complementarity determining regions (CDR) are indicated above the appropriate sequence segments in the figure.

Fig. 2

(A) Amino acid alignment of the V_H and V_L regions of the murine mAbs raised against SARS-CoV. The consensus sequence (top line) shows amino acid residues that are conserved in >50% of the sequences. A dot in the consensus sequence denotes amino acids that are conserved in <50% of the listed sequences. A dot in the individual sequences denotes amino acids that are the same as the consensus. A dash in the individual sequences denotes a deletion. Neutralizing mAbs are shown in bold text. The framework (FR) and complementarity determining regions (CDR) are indicated above the appropriate sequences in the figure. (B) Nucleotide alignments of identifiable D-regions in the V_H CDR3 regions. The most closely related D-regions were determined by V-quest analysis as outlined in Section 2. Dots represent identical residues, while dashes represent gaps or nucleotides that were not taken into account for the alignments. Neutralizing mAbs are shown in bold text.

Fig. 3

Summary of the percentage identity of the SARS-CoV-specific mAbs. (A) Summary of the percentage of pairwise identity, at the amino acid level (Y-axis), between the V_H chains of various categories of mAbs (all, neutralizing, non-neutralizing) as indicated on the X-axis. White bars: full length of sequences; hatched bars: CDR-1; grey bars: CDR-2; black bars: CDR-3. (B) Summary of the percentage of identity at the amino acid level of the V_L sequences. This figure is labeled the same as (A).

Fig. 4

Phylogenetic analysis of the mAbs specific for SARS-CoV. Trees are based on neighbor joining analysis of the inferred amino acid sequences of the relevant V_H and V_L sequences, as outlined in Section 2. (A) V_H regions of all mAbs. (B) V_H regions of all mAbs sharing significant identity with the J558.50 Ig germline V-gene. (C) V_L regions of all mAbs. In each case, the outliner sequences are of human origin, chosen as outlined in Section 2. Neutralizing mAbs are shown in bold text. The number at each node represents the level of bootstrap support (expressed as a percentage) for the node over the total number of replicates performed. Only those values above 50% are reported.