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Three key pathogens\u2014norovirus, rotavirus, and adenovirus 40\/41\u2014can induce dehydration, electrolyte imbalances, and severe complications, resulting in tens of thousands of deaths annually. Conventional vaccines have inherent limitations, including relatively long development cycles and high production costs. With the deep integration of bioinformatics and immunology, immunoinformatic techniques driven by high-throughput analysis enable reliable prediction of key epitope properties such as immunogenicity and antigenicity, offering an efficient approach for multivalent vaccine development. This study aims to develop a trivalent multi-epitope mRNA vaccine targeting these three pathogens using immunoinformatic methods, providing a potential innovative strategy for the prevention and control of diarrheal diseases.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>The amino acid sequences corresponding to the target viral proteins were obtained from the NCBI Virus Database. Epitopes were screened and selected based on key properties including high antigenicity, non-allergenicity, and non-toxicity. Appropriate adjuvant components, along with the chosen T-lymphocyte and B-lymphocyte epitopes, were assembled using linker molecules to computationally construct the vaccine. Structural and related features of the computationally designed vaccine were analyzed using online tools. Molecular docking assays, in conjunction with molecular dynamics simulations, were performed to clarify the interaction modes and structural stability characteristics of ligand-receptor binding. mRNA sequences of the vaccine were designed through codon optimization, and their immunogenicity was ultimately assessed using immune simulations.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>A total of 16 cytotoxic T-cell epitopes, 5 helper T-cell epitopes, and 17 linear B-cell epitopes were selected to construct the vaccine. After evaluating immunological and physicochemical properties, molecular docking and molecular dynamics simulations were performed, suggesting favorable structural stability and plausible interactions with immune receptors.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>The computationally designed vaccine in this study was predicted to exhibit favorable structural stability, potential immune activation capability, and promising broad population coverage, providing preliminary insights for the development of vaccines against multiple viral co-infections; however, its immunogenicity and safety remain to be further validated through animal model experiments.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s40249-026-01431-y","type":"journal-article","created":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T09:11:35Z","timestamp":1774602695000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Trivalent multi-epitope mRNA vaccine against norovirus, rotavirus, and adenovirus 40\/41: epitope screening, molecular docking, and molecular dynamics simulation with in silico validation guided by immunoinformatics"],"prefix":"10.1186","volume":"15","author":[{"given":"Xu","family":"Wu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yizhong","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rongliu","family":"Qin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuying","family":"Luo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanqun","family":"Hou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziyou","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruping","family":"Qu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiyang","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pinhua","family":"Pan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,3,27]]},"reference":[{"key":"1431_CR1","unstructured":"Estimates of the global, regional, and national morbidity, mortality, and aetiologies of diarrhoea in 195 countries: a systematic analysis for the global burden of disease study 2016. 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