HPV prophylactic vaccination based on VLPs was implemented 7 years ago and has now shown a high degree of efficiency to reduce HPV-induced lesions. Moreover, it was shown that HPV-derived virus-like particles or pseudovirions could be used as gene therapy vectors. As a consequence, characterization of the antigenic structure of HPV capsids is crucial for designing future HPV vaccines with better or broader efficacy and for the design of HPV-derived gene therapy vectors with reduced immunogenicity or vaccination escaping. In this study, we have generated 10 HPV16 FG loop L1 protein mutants and analyzed their ability to self-assemble into VLP, their immunogenicity, and their ability to transduce cells when used as pseudovirions. Most of the mutants had lost their ability to transduce cells at the exception of two chimeric HPV16/31 L1 protein FG loop mutants. Sera from mice immunized with HPV16 L1 wt VLPs very weakly neutralized pseudovirions derived from these two HPV16/31 L1 protein FG loop mutants. These findings suggest that only a few point substitutions within the FG loop are sufficient to generate a new serotype escaping vaccination. As a consequence, derived pseudovirions might be suitable as gene therapy vectors in vaccinated subjects.