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Viral myocarditis is a common clinical cardiovascular disease mainly induced by coxsackievirus B3 (CVB3) with no effective therapeutic measures. Induction of efficient mucosal immune responses is very critical against CVB3-induced myocarditis. FimH is an Escherichia coli (E. coli)-derived protein, which possesses an M cell-targeting property and functions as a TLR4 agonist. In this study, we introduced the recombinant FimH protein, into our previously developed CVB3 mucosal vaccine chitosan (CS)-pVP1, aiming to provoke more efficient mucosal immune responses and immunoprotection against CVB3-induced myocarditis. Compared with the CS-pVP1 vaccine, immunization with FimH-CS-pVP1 remarkably increased the levels and neutralizing titers of CVB3-specific protective secretory IgA (sIgA), enhanced the frequency of CVB3-specific IgA-producing B cells and amplified mucosal T-cell immune responses in mesenteric lymph nodes (MLNs), although failing to significantly amplify CVB3-specific systemic immune responses. Consistently, FimH-CS-pVP1 group showed the enhanced immunoprotection against CVB3-induced myocarditis, evidenced by the indices of limited myocardial injury, reduced viral loads and enhanced survival rate. Further study showed that this enhanced immunoprotection was not only ascribed to its M cell-targeting property, which led to the enhanced mucosal antigen VP1 expression, but also associated with the mucosal adjuvant effect of FimH, which facilitated the formation of germinal centers (GCs), production of IgA-inducing factors and maturation of antigen-presenting cells (APCs). Taken together, here we developed a bi-functional mucosal vaccine FimH-CS-pVP1, which simultaneously possessed the M cell-targeting property and mucosal adjuvant ability, and we showed that FimH-CS-pVP1 could efficiently induce the higher levels of CVB3-specific protective mucosal immune responses and provide better prophylactic effects against CVB3-induced myocarditis than CS-pVP1.

Efficient delivery of antigen to mucosal associated lymphoid tissue is a first and critical step for successful induction of mucosal immunity by vaccines. Considering its potential transcytotic capability, M cell has become a more and more attractive target for mucosal vaccines. In this research, we designed an M cell-targeting strategy by which mucosal delivery system chitosan (CS) was endowed with M cell-targeting ability via conjugating with a CPE30 peptide, C terminal 30 amino acids of clostridium perfringens enterotoxin (CPE), and then evaluated its immune-enhancing ability in the context of coxsackievirus B3 (CVB3)-specific mucosal vaccine consisting of CS and a plasmid encoding CVB3 predominant antigen VP1. It had shown that similar to CS-pVP1, M cell-targeting CPE30-CS-pVP1 vaccine appeared a uniform spherical shape with about 300 nm diameter and +22 mV zeta potential, and could efficiently protect DNA from DNase I digestion. Mice were orally immunized with 4 doses of CPE30-CS-pVP1 containing 50 μg pVP1 at 2-week intervals and challenged with CVB3 4 weeks after the last immunization. Compared with CS-pVP1 vaccine, CPE30-CS-pVP1 vaccine had no obvious impact on CVB3-specific serum IgG level and splenic T cell immune responses, but significantly increased specific fecal SIgA level and augmented mucosal T cell immune responses. Consequently, much milder myocarditis and lower viral load were witnessed in CPE30-CS-pVP1 immunized group. The enhanced immunogenicity and immunoprotection were associated with the M cell-targeting ability of CPE30-CS-pVP1 which improved its mucosal uptake and transcytosis. Our findings indicated that CPE30-CS-pVP1 may represent a novel prophylactic vaccine against CVB3-induced myocarditis, and this M cell-targeting strategy indeed could be applied as a promising and universal platform for mucosal vaccine development.