{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T04:27:07Z","timestamp":1787372827161,"version":"build-2736575974"},"reference-count":35,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2023YFD180250402"],"award-info":[{"award-number":["2023YFD180250402"]}]},{"DOI":"10.13039\/501100018927","name":"Southwest Minzu University","doi-asserted-by":"crossref","award":["ZYN2026026"],"award-info":[{"award-number":["ZYN2026026"]}],"id":[{"id":"10.13039\/501100018927","id-type":"DOI","asserted-by":"crossref"}]},{"award":["ZYN2026026"],"award-info":[{"award-number":["ZYN2026026"]}],"id":[{"id":"https:\/\/ror.org\/04gaexw88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Bovine coronavirus (BCoV) is a major cause of respiratory and enteric diseases in cattle, resulting in substantial economic losses to the global cattle industry. Next-generation epitope-focused vaccines require conserved neutralizing determinants to achieve broad protective efficacy across circulating variants. Here, we applied a reverse-vaccinology and immunoinformatics-guided strategy to identify conserved linear B-cell epitope candidates on the BCoV spike protein. Using ABCPred (16 aa; threshold score 0.51), 139 putative linear B-cell epitopes were predicted, and 10 candidates (B1\u2013B10) were selected based on antigenicity, allergenicity\/toxicity filters, and Shannon-entropy-based conservation (\u226595%). Each candidate was displayed on Helicobacter pylori ferritin and TEM confirmed the formation of self-assembled nanocages. BALB\/c mice (n = 6 per group) were subcutaneously immunized with 50 \u03bcg of each epitope\u2013ferritin fusion protein emulsified 1:1 with Montanide ISA 201 in a prime\u2013boost regimen (at a 14-day interval), and sera were collected 14 days after the booster immunization. All 10 constructs induced BCoV-specific binding IgG (endpoint titers ranging from 1:2667 to 1:11,733), but only B1, B2, B4, B6, B7, and B8 elicited detectable in vitro neutralizing activity against both representative strains, whereas B3, B5, B9, and B10 remained below the detection limit (&lt;1:8). Neutralizing titers were 1:24\u20131:99 for the enteric strain XHD4 and 1:27\u20131:88 for the respiratory strain HXD1, with no significant difference observed between the two strains (p &gt; 0.05). Overall, these findings identify six conserved B-cell epitope candidates capable of eliciting neutralizing antibody responses against both representative enteric and respiratory field strains, providing experimental evidence to support epitope-focused BCoV vaccine design.<\/jats:p>","DOI":"10.3390\/ani16162598","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:10:20Z","timestamp":1787213420000},"page":"2598","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of Conserved B-Cell Epitope Candidates Associated with Neutralizing Activity of Bovine Coronavirus Spike Protein"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7505-990X","authenticated-orcid":false,"given":"Yunxin","family":"Ren","sequence":"first","affiliation":[{"name":"Key Laboratory of Animal Medicine of Sichuan Education Department, Southwest Minzu University, Chengdu 610041, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Yue","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Medicine of Sichuan Education Department, Southwest Minzu University, Chengdu 610041, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng","family":"Tang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Medicine of Sichuan Education Department, Southwest Minzu University, Chengdu 610041, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Medicine of Sichuan Education Department, Southwest Minzu University, Chengdu 610041, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.vetmic.2015.10.017","article-title":"Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994\u20132010","volume":"181","author":"Bok","year":"2015","journal-title":"Vet. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Suzuki, T., Otake, Y., Uchimoto, S., Hasebe, A., and Goto, Y. (2020). Genomic Characterization and Phylogenetic Classification of Bovine Coronaviruses Through Whole Genome Sequence Analysis. Viruses, 12.","DOI":"10.3390\/v12020183"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Franzo, G., Drigo, M., Legnardi, M., Grassi, L., Pasotto, D., Menandro, M.L., Cecchinato, M., and Tucciarone, C.M. (2020). Bovine Coronavirus: Variability, Evolution, and Dispersal Patterns of a No Longer Neglected Betacoronavirus. Viruses, 12.","DOI":"10.3390\/v12111285"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1007\/s00705-012-1587-1","article-title":"Antigenic variation among recent Japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups","volume":"158","author":"Kanno","year":"2013","journal-title":"Arch. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/j.cvfa.2010.04.005","article-title":"Bovine Respiratory Coronavirus","volume":"26","author":"Saif","year":"2010","journal-title":"Vet. Clin. N. Am. Food Anim. Pract."},{"key":"ref_6","unstructured":"\u0110uri\u0107, M. (2022). Bovine Coronavirus Infection, CABI International."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3478","DOI":"10.1016\/j.vaccine.2016.04.020","article-title":"Detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease","volume":"34","author":"Fulton","year":"2016","journal-title":"Vaccine"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Biavasco, R., and De Giovanni, M. (2022). The relative positioning of B and T cell epitopes drives immunodominance. Vaccine, 10.","DOI":"10.1101\/2022.02.17.480954"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"De Groot, A.S., Moise, L., Terry, F., Gutierrez, A.H., Hindocha, P., Richard, G., Hoft, D.F., Ross, T.M., Noe, A.R., and Takahashi, Y. (2020). Better epitope discovery, precision immune engineering, and accelerated vaccine design using immunoinformatics tools. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.00442"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1016\/j.cell.2021.01.007","article-title":"Adaptive immunity to SARS-CoV-2 and COVID-19","volume":"184","author":"Sette","year":"2021","journal-title":"Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2921","DOI":"10.1038\/s41467-022-30658-0","article-title":"Antigenic structure of the human coronavirus OC43 spike reveals exposed and occluded neutralizing epitopes","volume":"13","author":"Wang","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Wang, C.Y., Peng, W.-J., Kuo, B.-S., Ho, Y.-H., Wang, M.-S., Yang, Y.-T., Chang, P.-Y., Shen, Y.-H., and Hwang, K.-P. (2023). Toward a pan-SARS-CoV-2 vaccine targeting conserved epitopes on spike and non-spike proteins for potent, broad and durable immune responses. PLoS Pathog., 19.","DOI":"10.1371\/journal.ppat.1010870"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1016\/j.meegid.2016.03.006","article-title":"Comparative molecular epidemiology of two closely related coronaviruses, bovine coronavirus (BCoV) and human coronavirus OC43 (HCoV-OC43), reveals a different evolutionary pattern","volume":"40","author":"Kin","year":"2016","journal-title":"Infect. Genet. Evol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Vlasova, A.N., and Saif, L.J. (2021). Bovine coronavirus and the associated diseases. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.643220"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Duraisamy, N., Khan, M.Y., Shah, A.U., Elalaoui, R.N., Cherkaoui, M., and Hemida, M.G. (2024). Machine learning tools used for mapping some immunogenic epitopes within the major structural proteins of the bovine coronavirus (BCoV) and for the in silico design of the multiepitope-based vaccines. Front. Vet. Sci., 11.","DOI":"10.3389\/fvets.2024.1468890"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5986893","DOI":"10.1155\/2024\/5986893","article-title":"Screening of bovine coronavirus multiepitope vaccine candidates: An immunoinformatics approach","volume":"2024","author":"Jiang","year":"2024","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1128\/CDLI.8.2.297-302.2001","article-title":"A single amino acid change within antigenic domain II of the spike protein of bovine coronavirus confers resistance to virus neutralization","volume":"8","author":"Yoo","year":"2001","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1002\/prot.21078","article-title":"Prediction of continuous B-cell epitopes in an antigen using recurrent neural network","volume":"65","author":"Saha","year":"2006","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"baac004","DOI":"10.1093\/database\/baac004","article-title":"GNIFdb: A neoantigen intrinsic feature database for glioma","volume":"2022","author":"Li","year":"2022","journal-title":"Database"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/nature12202","article-title":"Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies","volume":"499","author":"Kanekiyo","year":"2013","journal-title":"Nature"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Pattnaik, A., Sahoo, B.R., Struble, L.R., Borgstahl, G.E.O., Zhou, Y., Franco, R., Barletta, R.G., Osorio, F.A., Petro, T.M., and Pattnaik, A.K. (2023). A ferritin nanoparticle-based Zika virus vaccine candidate induces robust humoral and cellular immune responses and protects mice from lethal virus challenge. Vaccines, 11.","DOI":"10.3390\/vaccines11040821"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2202595","DOI":"10.1002\/adhm.202202595","article-title":"Engineered ferritin nanoparticle vaccines enable rapid screening of antibody functionalization to boost immune responses","volume":"12","author":"Vu","year":"2023","journal-title":"Adv. Healthc. Mater."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Langel, S.N., Paim, F.C., Alhamo, M.A., Buckley, A., Van Geelen, A., Lager, K.M., Vlasova, A.N., and Saif, L.J. (2019). Stage of gestation at porcine epidemic diarrhea virus infection of pregnant swine impacts maternal immunity and lactogenic immune protection of neonatal suckling piglets. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.00727"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"110313","DOI":"10.1016\/j.virol.2024.110313","article-title":"Isolation and molecular characterization of an enteric isolate of the genotype-Ia bovine coronavirus with notable mutations in the receptor binding domain of the spike glycoprotein","volume":"603","author":"Shah","year":"2025","journal-title":"Virology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/j.virol.2017.06.013","article-title":"The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes","volume":"509","author":"Okda","year":"2017","journal-title":"Virology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1126\/science.abj3321","article-title":"Broad betacoronavirus neutralization by a stem helix\u2013specific human antibody","volume":"373","author":"Pinto","year":"2021","journal-title":"Science"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1038\/s41594-021-00596-4","article-title":"Structural basis for broad coronavirus neutralization","volume":"28","author":"Sauer","year":"2021","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3210","DOI":"10.1021\/acs.chemrev.9b00472","article-title":"Peptide-based vaccines: Current progress and future challenges","volume":"120","author":"Malonis","year":"2019","journal-title":"Chem. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.smim.2017.08.014","article-title":"Major findings and recent advances in virus\u2013like particle (VLP)-based vaccines","volume":"34","author":"Mohsen","year":"2017","journal-title":"Semin. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1080\/14760584.2019.1578216","article-title":"Immunological basis for enhanced immunity of nanoparticle vaccines","volume":"18","author":"Kelly","year":"2019","journal-title":"Expert Rev. Vaccines"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"157895","DOI":"10.1155\/2014\/157895","article-title":"Neutralization of virus infectivity by antibodies: Old problems in new perspectives","volume":"2014","author":"Klasse","year":"2014","journal-title":"Adv. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1715","DOI":"10.1038\/s41467-021-21968-w","article-title":"A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies","volume":"12","author":"Wang","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"41931","DOI":"10.1074\/jbc.M112.418210","article-title":"Crystal structure of bovine coronavirus spike protein lectin domain","volume":"287","author":"Peng","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/bs.aivir.2016.08.004","article-title":"Coronavirus spike protein and tropism changes","volume":"96","author":"Hulswit","year":"2016","journal-title":"Adv. Virus Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.virusres.2014.11.021","article-title":"Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis","volume":"202","author":"Millet","year":"2015","journal-title":"Virus Res."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/16\/2598\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T04:12:37Z","timestamp":1787371957000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/16\/2598"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,20]]},"references-count":35,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ani16162598"],"URL":"https:\/\/doi.org\/10.3390\/ani16162598","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,20]]}}}