A rationally designed flagellin-L2 fusion protein induced serum and mucosal neutralizing antibodies against multiple HPV types

Abstract

The amino terminus of human papillomavirus (HPV) minor capsid protein L2 harbors several conserved neutralizing epitopes, including aa.17–36 (RG-1 epitope) and aa.65–85 consensus epitope (cL2 epitope), which are considered to be promising for the construction of cost-effective pan-HPV vaccine candidates. However, the immunogenicity of L2 epitope/peptide is rather weak, and the neutralizing spectrum induced by single type of L2 antigen is suboptimal. In this study, we constructed L2 concatemer with HPV18/33/58/59 RG-1 epitopes and 16L2 aa.11–88 peptide, and fused it with flagellin, a strong systemic and mucosal adjuvant, by hypervariable region replacement. A copy of cL2 epitope was also introduced to the C-terminus of the recombinant protein. The resultant Fla-5PcL2 protein can be produced in E. coli expression system with high yield and good stability. We assessed the immunogenicity of Fla-5PcL2 in mouse model via systemic and mucosal route, and found that subcutaneous immunization with Fla-5PcL2 induced robust serum neutralizing antibodies against divergent HPV types, while intranasal immunization with Fla-5PcL2 induced remarkable L2-specific IgA and cross-neutralizing antibodies in mucosal secretions, and medium titers of cross-neutralizing antibodies in sera. Moreover, Fla-5PcL2 induced full protection against vaginal HPV challenges. As mucosal antibodies provide the first-line defense at infection sites, and needle-free immunizations may increase vaccine compliance and require less public health resources, our results demonstrate that Fla-5PcL2 is a promising vaccine candidate which possibly meet the need in low-resource regions.