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A standardized protocol is needed to guide the process from gene selection to in silico optimization of the final vaccine construct. This protocol provides a comprehensive computational framework integrating all stages of mRNA vaccine design by defining the design space, essential criteria, and a step-by-step methodology. To demonstrate its applicability, the protocol is applied to the\n                    <jats:italic>SARS-CoV-2<\/jats:italic>\n                    spike protein, targeting both the coding sequence (CDS) and multi-epitope vaccine construct (MEVC). It incorporates immunoinformatics-based epitope selection, population coverage analysis, codon adaptation index optimization, minimum free energy-based RNA secondary structure assessment, target accessibility into a unified computational development workflow. The resulting constructs exhibited strong predicted immunogenicity and broad population coverage, further supported by in silico cloning and immune simulation validation for the optimized MEVC, consistent with prior in vitro and preclinical findings. Collectively, this protocol establishes a standardized computational roadmap for the design of CDS and MEVC mRNA vaccines, facilitating the development of stable, immunogenic, and translationally efficient vaccines against a wide range of pathogens.\n                  <\/jats:p>","DOI":"10.1186\/s13062-026-00846-9","type":"journal-article","created":{"date-parts":[[2026,6,13]],"date-time":"2026-06-13T11:33:23Z","timestamp":1781350403000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A protocol for computational design of mRNA vaccines with high functionality and specificity"],"prefix":"10.1186","volume":"21","author":[{"given":"Mennatallah A.","family":"Ramadan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Habiba M.","family":"ElGohary","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed HI","family":"Faraag","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed","family":"Mysara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,13]]},"reference":[{"issue":"5","key":"846_CR1","doi-asserted-by":"publisher","first-page":"479","DOI":"10.3390\/vaccines13050479","volume":"13","author":"S Boccalini","year":"2025","unstructured":"Boccalini S. 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