Characterization of the immune response to vibrio cholerae O1 antigens and its impact on vaccine development strategies

Abstract

Cholera persists as one of the major causes of mortality and morbidity globally. Prevention using different approaches are needed, one of which is vaccination. Improving immunogenicity of oral cholera vaccines and production of effective single dose schedules suitable for all age groups and populations is the prerequisite. Historically injectable cholera vaccines have been replaced in the last three decades by oral mucosal vaccines but neither fulfills all the requisites. The aim was to evaluate potential vaccine immunogens which have not been tested earlier for generating protective immunity. The concept of a one dose vaccine using bacterial antigen(s) through a “needle free approach” of immunization via the transcutaneous route (TCI) for enteric vaccines was explored in this work. The lipopolysaccharide (LPS) of Vibrio cholerae O1 is the most consistently described protection associated immunogen and has been identified as the most important of the antigens described so far. Epidemiological studies of natural infection in cholera suggest that Inaba serotype is more protective against subsequent attacks of cholera than the Ogawa serotype. Crystallographic studies have revealed antigenic determinants of LPS Ogawa and binding pattern of monoclonal antibodies (mAbs) using synthetic fragment of Ogawa in the O-specific polysaccharide (O-PS) region. Previous studies with other mAbs have indicated involvement of both the core and the O-specific polysaccharide as common antigenic determinants. This dissertation describes the first crystal structure of a mAb F-22-30 that recognizes a common epitope present in both the Ogawa and Inaba serotypes of V. cholerae O1. Modeling calculations and preliminary NMR results suggest that binding pocket at the center of antibody recombination site could bind side-chain of a perosaminyl residue from the O-PS with higher accessibility compared with core-PS implying a lateral recognition in contrast to antibodies specific for the Ogawa serotype. Mechanism of the immune response elicited by transcutaneous route of immunization (TCI) with potential V. cholerae antigens was evaluated as alternative modes of vaccination compared with oral (ORI) and subcutaneous route (SCI) with the same antigens i.e. recombinant cholera toxin B subunit (CTB), cholera toxin (CT), recombinant toxin co-regulated pilus A (TcpA), LPS Ogawa and Inaba and in-house prepared membrane extract in Balb/c mice to determine the immunoglobulin isotype patterns and its properties linked with protection. Both humoral and cellular immune responses induced by the immunization were studied using ELISA and flow cytometric procedures to assess and phenotypic changes in the mucosal and systemic compartments were analyzed. Among the three routes of immunization, SCI showed robust elevation pattern with superiority of IgG compared toIgM and IgA respectively. TcpA in the TCI route did not up-regulate IgA, while CT and CTB were seen to respond with all three antibody isotypes. CTB immunized mice showed reduced serum antibody titer in the zinc supplemented but CT and TcpA individually as well as co-administered together had no effect. When CT in the TCI and SCI route generated all four IgG subclasses, TcpA and LPS-specific antibody response was only IgG1 subclass where membrane extract has overcome the inherent limitation of LPS. However, sera obtained from both CT and TcpA immunized were negative for vibriocidal antibody responses. Membrane extract displayed more than 10 times elevated IgM and IgG titers with vibriocidal response than LPS with comparable positive effect of zinc supplementation. Moreover, ALS antibody titers were comparable with ELISA and vibriocidal assays and expressed the recently formed status of the humoral response. Expression of increased CD19+ve B cells in blood and lamina propria as well as CD4+ cells in the lamina propria were detected following TCI. Significant elevations of the co-stimulatory marker CD40 in the CD19+ve B cell population as well as CD28 and CD154 in the CD4 population were observed in spleen and lamina propria. The expression of homing markers, cutaneous lymphocyte antigen (CLA) was increased in blood and lamina propria (CD19, CD4), whereas an increase of the gut homing marker (β7) was not seen in blood or the mucosal surface in the different cell types. The results of TCI immunization were compared with those obtained after subcutaneous (SCI) where similar or discordant results were seen but interestingly the magnitude of responses by TCI was equal to that seen by SCI. The isotype switch and antibody maturation patterns elicited by the TCI route of immunization incorporating zinc as another confounder with the potential immunogens, applying a novel in-house immunogenetic method, were studied. Finally, bioinformatics approaches were applied to compare position specific similarities and dissimilarities of LPS specific 'membrane extract' immunized antibody sequences with experimentally characterized mAb F-22-30 for better understanding the binding affinity of the carbohydrate antigen. Considering the percentages of antigen-specific cellular representation at different compartments, CTB specific cells was the highest compared to TcpA and LPS specific pool. With the novel in situ positive selection immunogenetic technique, first murine B-cell VH and VL chain repertoire specific for protein and polysaccharide antigens were reported. VH and VL amino acid sequence comparison demonstrated variable expression in co-administration than individual immunogens. Amino acids expressed through immunization of polysaccharide immunogens were more stable than protein immunogens specific sequences. Moreover, CDRH3 length of antibody sequences to protein immunogens varied between 8 and 19 amino acids with CTB specific shorter sequences containing more charged and hydrophobic residues. Further bioinformatics approaches with Inaba specific sequences obtained through TCI confirmed ligand binding with similar nature amino acids to be identical groove type binding pattern. Findings presented here suggest that the newer, needle-free TCI route is suitable for inducing humoral and cellular immune responses in the systemic and mucosal compartments and should be considered as an option in vaccine development strategies.