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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-\u03b3. 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.<\/jats:p>","DOI":"10.34172\/bi.30943","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T11:48:17Z","timestamp":1784634497000},"page":"30943","update-policy":"https:\/\/doi.org\/10.34172\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Designing a novel therapeutic candidate vaccine for HPV16 using immunoinformatics approach: Targeting E6 and E7 epitopes with TLR4 agonist integration"],"prefix":"10.34172","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-5344-6278","authenticated-orcid":true,"given":"Zahra","family":"Samadi Moghaddam","sequence":"first","affiliation":[{"name":"Department of Medical Biotechnology, Maragheh University of Medical Sciences, Maragheh, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maryam","family":"Mashhadi Abolghasem Shirazi","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosa","family":"Jahangiri","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, Maragheh University of Medical Sciences, Maragheh, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Babak","family":"Negahdari","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8093-3297","authenticated-orcid":true,"given":"Nasser","family":"Hashemi Goradel","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, Maragheh University of Medical Sciences, Maragheh, Iran"},{"name":"Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"20123","published-online":{"date-parts":[[2026,5,16]]},"reference":[{"key":"R1","doi-asserted-by":"publisher","DOI":"10.31083\/j.fbl2810245"},{"key":"R2","doi-asserted-by":"publisher","DOI":"10.3322\/caac.21834"},{"key":"R3","doi-asserted-by":"publisher","DOI":"10.1007\/s10529-020-02792-6"},{"key":"R4","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2024.1362770"},{"key":"R5","doi-asserted-by":"crossref","unstructured":"Mlynarczyk-Bonikowska B, Rudnicka L. 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