Abstract In vaccinology, a potent immunogen has two prerequisite attributes—antigenicity and immunogenicity. We have rational designed a triple-type HPV vaccine against HPV58, −33 and −52 covered in Gardasil 9 based on the sequence homology and similar surface loop structure of L1 protein, which is related to cross-type antigenicity. Here, we design another triple-type vaccine against non-vaccine types HPV39, −68 and −70 by immunogenicity optimization considering type specific immunodominant epitopes located in separate region for different types. First, we optimized the expression of wild-type HPV39, −68 and −70 L1-only virus-like particles (VLPs) in E. coli through N-terminal truncation of HPV L1 proteins and non-fusion soluble expression. Second, based on genetic relationships and an L1 homologous loop-swapping rationale, we constructed several triple-type chimeric VLPs for HPV39, −68 and −70, and obtained the lead candidate named H39–68FG-70DE by the immunogenicity optimization using reactivity profile of a panel type-specific monoclonal antibodies. Through comprehensive characterization using various biochemical, VLP-based analyses and immune assays, we show that H39–68FG-70DE assumes similar particulate properties as that of its parental VLPs, along with comparable neutralization immunogenicity for all three HPV types. Overall, this study shows the promise and translatability of an HPV39/68/70 triple-type vaccine, and the possibility of expanding the type-coverage of current HPV vaccines. Our study further expanded the essential criteria on the rational design of a cross-type vaccine, i.e. separate sites with inter-type similar sequence and structure as well as type-specific immunodominant epitope to be clustered together.
