PLGA-entrapped recombinant r63 chimeric protein induces mucosal immunity against adherence and toxicity of Vibrio cholerae

Injectable inactivated whole-cell cholera vaccines provide limited protection due to poor mucosal immunity, leading to a shift toward oral cholera vaccines (OCVs). Chimeric recombinant vaccines can deliver multiple antigens simultaneously, enhancing immune specificity and simplifying production. This study aimed to develop a PLGA-encapsulated OCV using a chimeric protein for enhanced mucosal delivery and immune response. A synthetic gene encoding r63 was cloned into pET28a and expressed in E. coli BL21(DE3). The ~ 63 kDa His-tagged chimeric protein (r63) was purified via Ni-NTA affinity chromatography and entrapped in PLGA nanoparticles using a double emulsion-solvent evaporation technique. Particle size, zeta potential, SEM morphology, protein release kinetics, and encapsulation efficiency were evaluated. BALB/c mice were orally immunized with r63-PLGA, and IgG/IgA responses were measured by ELISA. Functional assays, including GM1-ELISA, Y-1 cell rounding, cAMP inhibition and rabbit ileal loop were performed to assess cholera toxin neutralization. In vitro adherence inhibition of V. cholera was investigated. The r63 was successfully expressed in E. coli and confirmed by SDS-PAGE and Western blotting. PLGA- r63 nanoparticles showed high encapsulation efficiency (90.16 ± 3.4%), a size of 221 ± 10 nm, and a zeta potential of - 11.8 ± 2 mV. Release studies indicated sustained protein release fitting a zero-order kinetic model. Oral immunization elicited robust serum IgG and fecal IgA responses against all antigens. Immunized sera prevented CTB binding to GM1 receptor, neutralized the enterotoxicity of cholera toxin in the Y1 cell line, and inhibited cAMP production. The immunized serum also suppressed cholera toxin activity in the ileal loop test by up to 79% and inhibited 66% of the bacterial adhesion to Caco2 cells. The orally delivered PLGA-r63 candidate vaccine effectively induced systemic and mucosal immunity against adherence and toxicity Vibrio cholerae.