An improved claudin-targeting mucosal vaccine using a double alanine-substituted mutant of the Cterminal fragment of Clostridium perfringens enterotoxin

H. Suzuki1, J. Kunisawa2, 3, A. Watari1, M. Yamashita1, K. Yagi1 and M. Kondoh1
1Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan; 2Laboratory of Vaccine Materials, National Institute of Biomedical Innovation, Osaka 567-0085, Japan; 3International Research and Development Center for Mucosal Vaccine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan


Efficient delivery of antigen to mucosa-associated lymphoid tissues (MALT) is essential for mucosal vaccination. Claudin-4 (CL-4), a key structural and functional component of the tight junction, is expressed on the epithelial cells that cover MALT. We previously found that CL-4 targeting by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a CL-4 binder is an effective strategy for the development of mucosal vaccines. Deletion of the 10 N-terminal amino acids of C-CPE increased its solubility, and substitution of Asn309 and Ser313 with Ala increased the affinity of C-CPE for CL-4. Here, we investigated whether a C-CPE mutant lacking the 10 terminal amino acids and carrying Ala309 and Ala313 could serve as a mucosal vaccine. We fused this Ala-substituted C-CPE mutant with ovalbumin (OVA) as a model antigen to generate the OVA-C-CPE mutant construct. Intranasal immunization with OVA-C-CPE mutant increased the production of OVA-specific serum IgG and mucosal IgA compared with that in mice immunized with OVA-C-CPE. In addition, immunization with OVA-C-CPE mutant activated both Th1 and Th2 responses and induced anti-tumor activity in mice inoculated with OVA-expressing thymoma cells. These findings suggest that the Ala-substituted C-CPE mutant is a promising CL-4 binder for claudin-targeted mucosal vaccination.

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