摘要Objective:Cervical cancer caused by persistent high-risk human papillomavirus(hrHPV)infection remains a leading cause of cancer-related mortality in women.As prophylactic HPV vaccines cannot eliminate existing infections,developing therapeutic vaccines targeting HPV E6/E7 oncoproteins is critical for reversing precancerous lesions.This study aimed to design a novel multi-epitope vaccine against HPV16,incorporating newly identified immunodominant epitopes and evaluating the therapeutic efficacy.Methods:The multi-epitope vaccine HSP70-12P was bioinformatically designed to include cytotoxic T lymphocyte(CTL)and helper T lymphocyte(HTL)epitopes from HPV16 E6/E7,which were fused to the C-terminal domain(residues 359-610)of Mycobacterium tuberculosis HSP70 as an adjuvant.Two formulations were used,as follows:(1)protein-based Pro-HSP70-12P;and(2)DNA-based DNA-HSP70-12P.Therapeutic efficacy was evaluated in TC-1 tumor-bearing mouse models.Tumor regression,survival rates,and immune correlates(T cell responses and cytokine profiles)were assessed.Immunodominant epitopes were identified using ELISpot.Results:The Pro-HSP70-12P protein vaccine induced strong immune responses and provided lasting antitumor protection.The vaccine activated cell-mediated immunity and stimulated effector memory T cells in the HPV-16-related tumor mouse model,resulting in strong tumor clearance effects.Pro-HSP70-12P demonstrated superior performance compared to the DNA-HSP70-12P vaccine,achieving complete regression of small tumors(diameter<2 mm)with a single dose and conferring long-lasting protection in TC-1 rechallenge experiments.Three novel immunodominant epitopes were identified(E6-38-45,E6-124-132,and E7-50-57).The E6 epitopes address a critical gap in E6-targeted vaccine design.Conclusions:The multi-epitope protein vaccine,Pro-HSP70-12P,represents a potent therapeutic candidate against HPV-driven malignancies,which has the capacity to induce tumor regression and long-term immunity.These findings support further clinical development.