Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

Abstract

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.