Construction, mucosal immunogenicity and in vitro protective efficacy of a genetically engineered pertussis toxin-cholera toxin chimeric protein against Bordetella pertussis

Induction of a protective antibody immune response at mucosal surfaces, the initial barrier to most pathogens, is not readily achieved by systemic or mucosal administration of vaccine antigens. In this work, the capacity of cholera toxin A2/B (CtxA2/B) to act as a carrier for mucosal delivery of vaccine antigens was exploited by constructing a genetic fusion consisting of DNA encoding CtxA2/B cloned downstream of two in tandem copies of DNA encoding the 179 amino acid fragment of N-terminus pertussis toxin S1. This fusion was downstream of DNA encoding a Streptococcus mutans spaP promoter and 561 SpaP N-terminal amino acids creating a spaP/ s1/s1/ctxA2/B operon. The s1/s1/ctxA2/ B fragment was subsequently cloned downstream of the maltose-binding protein (MBP) gene in pMALp. In-frame fusion was demonstrated by Western blotting and GM 1-binding ELISA. Expression of MBP/S1/S1/CtxA2/B was induced by IPTG and the chimeric protein was solubilized and isolated using 6 M urea, as well as column chromatography. SDS-PAGE and Western blotting confirmed isolation of the chimeric protein. GM1-binding ELISA demonstrated that the fusion protein is associating with the CtxB-pentamer, presumably via the A2 fragment, forming the desired macromolecule. Intranasal administration of the MBP/S1/S1/CtxA2/B chimera induced a mucosal (salivary IgA) and a systemic immune (serum IgG) response to PT and CtxB in female BALB/c mice. Sera from the chimera immunized mice neutralized the cytotoxic effect of PT on Chinese hamster ovary (CHO) cells. In conclusion, a divalent pertussis toxin S1 fragment was successfully fused to cholera toxin A/2B moiety and the chimeric protein, purified from Escherichia coli, induces a mucosal and systemic immune response, the latter of which was demonstrated to neutralize the cytotoxic effects of pertussis toxin on Chinese hamster ovary cells.