Single cell analyses of human antibody responses to Vibrio cholerae

Abstract

Abstract Cholera is a severe diarrheal disease affecting 2.9 million people annually resulting in an estimated 95,000 deaths. Infection induces long lasting protective immunity, likely mediated by intestinal antibodies. To further understand humoral immunity against cholera, we used single cell expression cloning to generate panels of human monoclonal antibodies (mAbs) from acutely induced plasmablasts isolated from patients in Bangladesh. mAbs largely targeted the dominant antigens cholera toxin and lipopolysaccharide (LPS). Notably, while LPS responses targeted the O-specific polysaccharide moiety, mAbs varied in serotype specificity and functional characteristics. Unexpectedly, despite all patients being infected by the Ogawa serotype, isolated mAbs from one patient preferentially bound to the Inaba serotype, which had been almost undetectable in circulation for 5 years. Inaba selective mAbs were characterized by high levels of somatic hypermutation. These findings suggest that cholera can generate immunologic memory and induce significant somatic hypermutation in response to this polysaccharide antigen. To expand on these findings we generated a panel of isotype and subclass variants from six LPS specific mAbs, representing a range of affinity and serotype specificity. mAbs were expressed as IgG1, IgG2, IgG3, IgG4, pentameric IgM, and the monomeric and dimeric forms of IgA1 and IgA2. This analysis provided detailed insight into how immunoglobulin isotype/subclass impacts functional characteristics such as agglutination and vibriocidal activity. Moreover, using live-cell microscopy and soft-agar migration assays, we provide novel insight into the impact of these antibody variants on bacterial propulsion.