Generation and Characterization of Monoclonal Antibodies to the Ogawa Lipopolysaccharide of Vibrio cholerae O1 from Phage-Displayed Human Synthetic Fab Library

Abstract

Vibrio cholerae , cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2- O -methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.

Generation of Single‐Chain Variable Fragment (scFv) Libraries for Use in Phage Display

“…Before beginning the experiments listed, careful consideration should be given to the type of protein or peptide to be generated and displayed on the phage surface. There are several options available, such as scFvs, described here; antigen‐binding fragments (Fabs; Kim et al., 2020); nanobodies (Rossey et al., 2017); and cyclic peptides (Zhai et al., 2020). Depending on the chosen product, the cloning strategy detailed in these protocols may not be applicable, which will necessitate further primer design to ensure generation of the desired product.…”