{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T09:24:32Z","timestamp":1787217872396,"version":"build-2736575974"},"reference-count":46,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Therapeutic vaccination against human papillomavirus type 16 (HPV16) is frequently limited by the weak HLA-A*02:01 presentation of native E6\/E7 oncoprotein epitopes. This study aims to utilize an integrated computational-experimental pipeline to design and evaluate anchor-optimized altered peptide ligands (APLs) for improving peptide presentation and HPV16-specific T-cell responses. Methods: Anchor-residue substitutions were introduced into three wild-type E6\/E7 epitopes. Candidates were prioritized in silico based on predicted presentation, affinity, immunogenicity, and toxicity, and subsequently evaluated via in vitro functional assays using HLA-A*02:01-positive donor cells and molecular dynamics (MD) simulations. Results: Anchor optimization successfully converted weak binders into strong binders; for instance, E6apl improved predicted affinity from 329.33 to 6.21 nM. Crucially, candidate E7apl1 exhibited normal CD8+ T-cell expansion but reduced IFN-\u03b3 secretion, revealing a distinct binding\u2013immunogenicity dissociation. MD simulations suggested that altered peptide conformational dynamics may contribute to differences in functional activity. Subsequently, an optimized six-peptide formulation (three APLs and three wild-type epitopes) was assembled. The resulting multi-epitope HPV-specific cytotoxic T lymphocytes (meHPV-CTLs) mediated target-specific cytotoxicity against cervical cancer cells, achieving 74.1% \u00b1 5.1% specific lysis at an effector-to-target ratio of 30:1, which was largely abrogated by HLA class I blockade. Conclusions: These proof-of-concept findings demonstrate that stable peptide\u2013MHC binding is a necessary but insufficient condition for optimal T-cell activation. The experimentally characterized multi-epitope formulation provides a proof-of-concept strategy for further preclinical evaluation of HPV16-targeted peptide-based immunotherapies. Moreover, because these anchor-optimized APLs are defined at the sequence level, these sequence-defined APLs may potentially be explored in alternative vaccine delivery platforms, including mRNA-based approaches, in future studies.<\/jats:p>","DOI":"10.3390\/vaccines14080715","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:10:20Z","timestamp":1787213420000},"page":"715","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Immunoinformatics-Driven Rational Design of Altered Peptide Ligands and In Vitro Validation of a Multi-Epitope CTL Formulation Targeting HPV16 E6\/E7"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-1829-8263","authenticated-orcid":false,"given":"Dian","family":"Dong","sequence":"first","affiliation":[{"name":"College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China"},{"name":"BGI Research, Shenzhen 518083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuqing","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China"},{"name":"BGI Research, Shenzhen 518083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Li","sequence":"additional","affiliation":[{"name":"BGI Research, Shenzhen 518083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","first-page":"209","article-title":"Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. 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