Impact of Immunoglobulin Isotype and Epitope on the Functional Properties ofVibrio choleraeO-Specific Polysaccharide-Specific Monoclonal Antibodies

Abstract

Vibrio cholerae causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition. IMPORTANCE Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.

Interactions between the Prophage 919TP and Its Vibrio cholerae Host: Implications of gmd Mutation for Phage Resistance, Cell Auto-Aggregation, and Motility

“…This is in agreement with a previous finding, in which a waaL mutant that lacks O-antigen also had impaired motility, affecting the ability of V. fischeri to spread in soft agar [35]. In addition, LPS-specific, antibody-mediated motility arrest, at the level of the individual V. cholerae bacterium, has been demonstrated to effectively inhibit cholera dispersal or provide protection in vivo [36]. O-antigen is considered a major virulence factor that is required, for instance, in vertical localization, small intestine colonization, and resistance to the immune response and antibiotics [32].…”

Scalable production and immunogenicity of a cholera conjugate vaccine

“…OSP-specific antibody responses inhibit V. cholerae motility, and this effect may be mechanistically involved in protection against cholera within the intestinal lumen of infected humans [41] . Blocking the ability of the highly mobile and freely-swimming V. cholerae pathogens from reaching their target ecological niche within the intestinal lumen of infected humans and delivering cholera toxin to intestinal epithelial cells may be protective [41] , [42] .…”

Systemic, Mucosal, and Memory Immune Responses following Cholera

“…Much effort is now being made to assess how OSP-specific antibodies might protect against cholera, with a growing body of evidence suggesting that protection against infection may involve the ability of OSP-specific antibodies to impede the motility of V. cholerae organisms in the human intestine. This effect requires at least two-point binding of OSP-specific antibodies [ 21 , 22 , 23 , 24 , 25 , 26 ].…”

“…There is no evidence that enhanced opsonophagocytosis or antibody-dependent cytotoxic activity in the intestinal lumen plays a role in mediating protection against cholera. Although cell-free antibody-based killing via complement lysis might be considered possible in the intestinal lumen, viability studies in animals have shown that bactericidal activity is not required for protection from disease [ 22 , 25 , 26 ]. In addition, although C3 and earlier components of the complement cascade have been detected in the intestinal lumen/epithelium, the terminal components of the complement cascade have not been detected in the lumen in the absence of epithelial breakdown and intestinal inflammation [ 38 , 39 , 40 ].…”

“…Mucosal IgA and IgM antibodies may protect against pathogens at mucosal surfaces by inhibiting bacterial-epithelial cell interactions (“immune exclusion”), and/or via bacterial “clumping” either via agglutination at high bacterial concentrations (≥10 8 non-motile bacteria per gram), or “enchaining growth” (antibody-mediated cross-linking preventing bacterial separation after bacterial division) at lower bacterial concentrations, with facilitated mucosal clearance of clumped bacteria [ 55 ]. Recent data also suggest that inhibition of motility of V. cholerae in the intestinal lumen may contribute mechanistically to protection against cholera [ 25 , 26 ].…”