Tabriz University of Medical Sciences BioImpacts 2228-5652 16 1 2026 01 04 Designing a novel therapeutic candidate vaccine for HPV16 using immunoinformatics approach: Targeting E6 and E7 epitopes with TLR4 agonist integration 30943 30943 10.34172/bi.30943 EN Zahra Samadi Moghaddam https://orcid.org/0009-0006-5344-6278 Maryam Mashhadi Abolghasem Shirazi Rosa Jahangiri Babak Negahdari Nasser Hashemi Goradel https://orcid.org/0000-0001-8093-3297 Journal Article 10.34172/bi.30943 2024 12 25 2026 02 18 Introduction: Although prophylactic vaccinations for human papillomaviruses (HPVs) have been approved, these vaccines lack therapeutic efficacy and cannot eradicate pre-existing infections. Although epitope-based vaccines represent a promising therapeutic vaccine platform, their anti-tumor efficacy has been limited due to low immunogenicity. This study aimed to apply bioinformatics tools to design a built-in adjuvant therapeutic candidate vaccine targeting HPV16 infections and associated cancers. Methods: The designed vaccine consists of HPV16 E6 and E7 epitopes conjugated to the domain 4 of pneumolysin (Ply4) from Streptococcus pneumonia, which serves as a potential toll-like receptor 4 (TLR4) agonist. In silico analyses were performed to evaluate the vaccine's physicochemical properties, antigenicity, immunogenicity, and binding interactions with the TLR4 receptor. The designed vaccine was expressed in E. coli and its expression was confirmed by SDS-PAGE and Western blot analysis. Results: In silico analysis predicted that the designed vaccine could have desirable qualities, including non-toxicity, non-allergenicity, antigenicity, immunogenicity, hydrophilicity, and stability. Docking analysis between the vaccine and the TLR4 proteins predicted a high binding capacity and efficient binding. Furthermore, immunoinformatics tools showed that the vaccine could induce robust immune responses, specifically helper and cytotoxic T-cell responses, and promote the production of IFN-γ. The vaccine was successfully expressed in the E. coli system after being cloned into the pET28a vector. SDS-PAGE and Western blotting assays confirmed the purification of the target protein. Conclusion: The novel built-in adjuvant therapeutic candidate vaccine is a rationally designed construct for eradicating pre-existing HPV infections and HPV-induced cervical cancers that warrants further preclinical evaluation.