{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T21:55:10Z","timestamp":1782251710976,"version":"3.54.5"},"reference-count":93,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T00:00:00Z","timestamp":1766016000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>Background: Tuberculosis (TB) remains a pressing global health crisis. The inadequate efficacy of the BCG vaccine against adult pulmonary TB underscores the urgent need for novel, effective vaccines. This study aimed to design a novel mRNA vaccine candidate against TB using a rational immunoinformatics approach. Methods: From 13 antigens, &gt;12,000 epitopes were filtered to select 60 optimal peptides (36 CTL, 16 HTL, 8 B-cell), assembled into 25 scaffolds with 49 TLR2\/4 agonist configurations. EP9158H underwent structural modeling, 100 ns molecular dynamics, docking, immune simulation, RNAfold, and conservation analysis across 76 strains. Results: EP9158H, encoding 15 CTL, 9 HTL, and 8 B-cell epitopes flanked by TLR2 agonist ESAT-6 and TLR4 agonist HBHA, emerged as the optimal candidate. All 32 constituent epitopes showed &gt;81% conservation, with 81.25% exhibiting perfect identity across MTBC lineages. The scaffold demonstrated high solubility (0.531), broad population coverage (73.76% MHC-I, 88.91% MHC-II), optimal TLR2\/4 docking scores (\u22121359.7 and \u22121348.3), and robust structural stability (ProSA Z-score \u22126.18; RMSD 22\u201327 \u00c5). Immune simulation predicted strong Th1-biased T-cell responses and high levels of antibody titers. RNAfold analysis revealed stable mRNA secondary structures (MFE \u22121127.5 kcal\/mol) supporting efficient translation. Conclusions: EP9158H integrates broad epitope coverage, dual TLR agonism, and validated stability. Compared to single-antigen vaccines, it offers superior strain coverage, enhanced innate activation, and mRNA advantages for CTL induction, warranting experimental validation.<\/jats:p>","DOI":"10.3390\/bioengineering12121378","type":"journal-article","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T14:32:03Z","timestamp":1766068323000},"page":"1378","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["EP9158H: An Immunoinformatics-Designed mRNA Vaccine Encoding Multi-Epitope Antigens and Dual TLR Agonists for Tuberculosis Prevention"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7229-5242","authenticated-orcid":false,"given":"Mingming","family":"Zhang","sequence":"first","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"},{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Syed Luqman","family":"Ali","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Abdul Wali Khan University, Mardan 23200, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7407-3126","authenticated-orcid":false,"given":"Yuan","family":"Tian","sequence":"additional","affiliation":[{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aigul","family":"Abduldayeva","sequence":"additional","affiliation":[{"name":"Department of Research Institute of Preventive Medicine Named Academician E. Dalenov, Astana Medical University, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-4940-5061","authenticated-orcid":false,"given":"Shuang","family":"Zhou","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"},{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"},{"name":"Department of Geriatrics, The Eighth Medical Center of PLA General Hospital, Beijing 100091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7016-0008","authenticated-orcid":false,"given":"Yajing","family":"An","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"},{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2483-1267","authenticated-orcid":false,"given":"Yufeng","family":"Li","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"},{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"},{"name":"Department of Geriatrics, The Eighth Medical Center of PLA General Hospital, Beijing 100091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2522-0257","authenticated-orcid":false,"given":"Ruizi","family":"Ni","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"},{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6596-2847","authenticated-orcid":false,"given":"Lingxia","family":"Zhang","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8095-3930","authenticated-orcid":false,"given":"Yanhua","family":"Liu","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiguo","family":"Sun","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0333-890X","authenticated-orcid":false,"given":"Wenping","family":"Gong","sequence":"additional","affiliation":[{"name":"Senior Department of Tuberculosis, Chinese PLA General Hospital, Beijing 100091, China"},{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e70054","DOI":"10.1002\/imt2.70054","article-title":"Gut microbiota and tuberculosis","volume":"4","author":"Liu","year":"2025","journal-title":"iMeta"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.15212\/ZOONOSES-2024-0061","article-title":"Decoding the WHO Global Tuberculosis Report 2024: A Critical Analysis of Global and Chinese Key Data","volume":"5","author":"Chen","year":"2025","journal-title":"Zoonoses"},{"key":"ref_3","first-page":"e56643","article-title":"Bacillus Calmette-Gu\u00e9rin (BCG) Revaccination and Protection Against Tuberculosis: A Systematic Review","volume":"16","author":"Lawrence","year":"2024","journal-title":"Cureus"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s43556-024-00243-6","article-title":"Tuberculosis vaccines and therapeutic drug: Challenges and future directions","volume":"6","author":"An","year":"2025","journal-title":"Mol. 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