{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T12:11:15Z","timestamp":1789128675467,"version":"build-2803163510"},"reference-count":56,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Usutu virus is an emerging mosquito-borne flavivirus with an expanding geographic distribution and increasing public health relevance, yet no licensed vaccine is currently available. This study used an integrated reverse vaccinology strategy to identify conserved immunogenic regions from the Envelope protein and NS5 protein, and construct a multi-epitope vaccine. Following sequential computational screening, the retained T-cell and B-cell epitopes satisfied the predefined selection criteria, while selected T-cell epitopes achieved an estimated 96.41% global population coverage. The final vaccine consisted of 240 amino acids and incorporated an adjuvant together with peptide linkers. Computational characterization indicated favorable physicochemical features and a refined three-dimensional model with improved stereochemical characteristics. Receptor-binding analyses predicted favorable interactions with TLR2 and TLR4, producing weighted docking scores of \u22121326.1 and \u22121230.2, respectively. Molecular dynamics simulation further characterized the temporal behavior of the vaccine\u2013TLR2 complex, while MM-GBSA analysis yielded an estimated binding energy of \u221274.78 kcal\/mol. C-ImmSim predicted enhanced humoral and cellular immune-response patterns following repeated antigen administration, including increased simulated antibody levels and changes in immune-cell populations. All findings in this study are based on in silico analyses and represent computational predictions rather than experimentally confirmed results. Further experimental validation is required to verify the predicted properties, immunogenicity, and protective potential of the proposed vaccine candidate.<\/jats:p>","DOI":"10.3390\/microorganisms14092026","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T10:51:37Z","timestamp":1789123897000},"page":"2026","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Design and Evaluation of a Multi-Epitope Vaccine Targeting Conserved Envelope and NS5 Proteins of Usutu Virus Using Immunoinformatics"],"prefix":"10.3390","volume":"14","author":[{"given":"Reem","family":"Alromaihi","sequence":"first","affiliation":[{"name":"Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4566-2932","authenticated-orcid":false,"given":"Hajed Obaid","family":"Alharbi","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8330-7945","authenticated-orcid":false,"given":"Suleman Abdullah","family":"Almerdasi","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9550-7150","authenticated-orcid":false,"given":"Mawahib A.","family":"Ahmed","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8619-0081","authenticated-orcid":false,"given":"Waad A.","family":"Aljohani","sequence":"additional","affiliation":[{"name":"Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mona","family":"Alromaihi","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, College of Medicine, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laila","family":"Alhussain","sequence":"additional","affiliation":[{"name":"Department of Biology, College of Science, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alaa","family":"Karkashan","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, College of Science, University of Jeddah, Jeddah 21959, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riham Mohamad","family":"Rashad Mohamad","sequence":"additional","affiliation":[{"name":"Department of Laboratory and Blood Bank, Buraydah Central Hospital, Buraydah 52361, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6486-9835","authenticated-orcid":false,"given":"Khaled S.","family":"Allemailem","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Akinsulie, O.C., Adesola, R.O., Bakre, A., Adebowale, O.O., Adeleke, R., Ogunleye, S.C., and Oladapo, I.P. 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