Evolutionary Dynamics of Human Papillomavirus in Thailand: Immunoinformatic Design of a Synergistic L1/L2 Vaccine Candidate

Background/Objectives: Current prophylactic human papillomavirus (HPV) vaccines rely on major capsid proteins (L1). Although highly immunogenic, L1-based immunity is clade-restricted owing to the hypervariability of HPV strains across different geographic regions. This restricts cross-protection against diverse, localized, and non-vaccine viral strains circulating in specific areas, leaving these populations vulnerable to regional genotypes. Methods: We aimed to design a cross-protective vaccine tailored to local viral diversity through evolutionary and immunoinformatic analyses of circulating HPV strains in Thailand. Both major (L1) and minor (L2) capsid proteins were analyzed to evaluate selective evolutionary pressures, structural sequence conservation, and cross-strain promiscuity of predicted B- and T-cell epitopes using the IEDB database. The predicted immunogenic profile of the combined L1/L2 proteins was further assessed using in silico immune response simulations. Results: Evolutionary analysis confirmed that although the L1 protein is under strict purifying selection, it exhibits high regional interclade variability. L1-only epitopes demonstrated restricted cross-strain conservation, creating structural blind spots against divergent regional variants. The minor capsid protein (L2) harbored highly conserved dual-action core peptides that are evolutionarily conserved across multiple HPV type. Incorporation of conserved L2 regions alongside L1 sequences may broaden the predicted epitope coverage and help address limitations associated with L1 variability. The proposed multi-targeted L1/L2 construct therefore represents a computational framework for the development of candidate cross-reactive HPV vaccines relevant to circulating Thai genotypes. However, experimental validation remains necessary to determine immunogenicity, cross-neutralization potential, and protective efficacy. Conclusion: Our findings highlight these conserved HPV epitopes as potential targets for future development of next-generation cross-subtype prophylactic vaccines with broader population coverage.