{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T06:43:03Z","timestamp":1791614583472,"version":"4.3.4"},"reference-count":143,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T00:00:00Z","timestamp":1771977600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      The parasite\n                      <jats:italic>Cryptosporidium<\/jats:italic>\n                      spp. causes cryptosporidiosis, a diarrheal disease in humans and animals. This study describes the development of mRNA vaccine targeting antigens from\n                      <jats:italic>C. hominis<\/jats:italic>\n                      and\n                      <jats:italic>C. parvum<\/jats:italic>\n                      , important gut pathogens. This vaccine was designed with reverse vaccinology and immunoinformatics as no FDA-approved vaccine exists for cryptosporidiosis.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Materials and methods<\/jats:title>\n                    <jats:p>Initially, a thorough literature review was conducted to identify five pathogenic proteins (aminopeptidase, heat shock protein, P23, serine protease, and sporozoite glycoproteins) associated with these two parasites. Next, a multiple sequence alignment was conducted, and the conserved sequences were used to design a novel multiepitope mRNA vaccine against these two parasites, combining the best CD8+, CD4+, and continuous B-cell epitopes. Additionally, structural prediction, docking, dynamics, and immune simulation, as well as cloning, were conducted.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Result<\/jats:title>\n                    <jats:p>\n                      The vaccine demonstrated standard biophysical properties, indicating that the protein is soluble and stable. Both two-dimensional (substantial alpha helix, beta sheet, and coil structures) and three-dimensional structures (Ramachandran score of 83.1% and a Z score of \u22127.39) of the vaccine were standard. The docking energy for TLR-2 (\u22121151.9) and TLR-4 (\u22121028.3) exhibited significant interactions. Furthermore, MM-GBSA and dynamics simulation both verified their stability, compactness, and flexibility. Next, codon optimization for\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      expression yielded promising results, with the vaccine demonstrating substantial expression, as evidenced by a GC content of 46.97% and a CAI of 0.988. Afterwards, immune simulation demonstrated robust immune response amplification upon repeated exposures. In addition, the vaccine exhibited stability in its mRNA structures.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      This study developed an\n                      <jats:italic>i<\/jats:italic>\n                      <jats:italic>n-silico<\/jats:italic>\n                      multiepitope novel mRNA vaccine candidate for\n                      <jats:italic>C. hominis<\/jats:italic>\n                      and\n                      <jats:italic>C. parvum<\/jats:italic>\n                      with excellent structural stability, antigenicity, receptor-binding affinity, and expected immune responses. These findings offer a novel approach due to numerous species target but with significant drawbacks like no validation beyond simulation, uncertainty of long-term immunity, protein quality, stability and safety, requiring experimental validation.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pone.0343643","type":"journal-article","created":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T18:31:22Z","timestamp":1772044282000},"page":"e0343643","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["A novel mRNA-based multiepitope vaccine candidate against Cryptosporidium hominis and Cryptosporidium parvum employing reverse-vaccinology and immunoinformatics approaches"],"prefix":"10.1371","volume":"21","author":[{"given":"Ahmad Abdullah","family":"Mahdeen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2516-8689","authenticated-orcid":true,"given":"Imam","family":"Hossain","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0055-1895","authenticated-orcid":true,"given":"Md. Habib Ullah","family":"Masum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"T. M. Fazla","family":"Rabbi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sajedul","family":"Islam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,2,25]]},"reference":[{"issue":"1","key":"pone.0343643.ref001","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/s12223-012-0182-x","article-title":"The first reported cases of human cryptosporidiosis caused by Cryptosporidium hominis in Slovak Republic","volume":"58","author":"F Ondriska","year":"2013","journal-title":"Folia Microbiol (Praha)"},{"issue":"10","key":"pone.0343643.ref002","doi-asserted-by":"crossref","first-page":"1567","DOI":"10.3201\/eid1410.071273","article-title":"Cryptosporidium species and subtypes and clinical manifestations in children, Peru","volume":"14","author":"VA Cama","year":"2008","journal-title":"Emerg Infect Dis"},{"issue":"1","key":"pone.0343643.ref003","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1128\/CMR.17.1.72-97.2004","article-title":"Cryptosporidium taxonomy: recent advances and implications for public health","volume":"17","author":"L Xiao","year":"2004","journal-title":"Clin Microbiol Rev"},{"issue":"4","key":"pone.0343643.ref004","doi-asserted-by":"crossref","first-page":"882","DOI":"10.2478\/s11686-020-00230-1","article-title":"Prevalence of Cryptosporidium Infection in the Global Population: A Systematic Review and Meta-analysis","volume":"65","author":"S Dong","year":"2020","journal-title":"Acta Parasitol"},{"issue":"12","key":"pone.0343643.ref005","doi-asserted-by":"crossref","first-page":"2426","DOI":"10.3390\/microorganisms12122426","article-title":"Cryptosporidium Species Infections Detected from Fecal Samples of Animal and Human Hosts in South Africa: Systematic Review and Meta-Analysis","volume":"12","author":"M Tawana","year":"2024","journal-title":"Microorganisms"},{"issue":"22","key":"pone.0343643.ref006","doi-asserted-by":"crossref","first-page":"1723","DOI":"10.1056\/NEJMra013170","article-title":"Cryptosporidiosis","volume":"346","author":"X-M Chen","year":"2002","journal-title":"N Engl J Med"},{"key":"pone.0343643.ref007","unstructured":"CDC. 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