{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,28]],"date-time":"2026-10-28T03:45:35Z","timestamp":1793159135814,"version":"4.5.0"},"reference-count":88,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2022,7,18]],"date-time":"2022-07-18T00:00:00Z","timestamp":1658102400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Agricultural Science and Technology Cooperation Program of Zhejiang Province","award":["2022SNJF060"],"award-info":[{"award-number":["2022SNJF060"]}]},{"name":"Agricultural Science and Technology Cooperation Program of Zhejiang Province","award":["2022C02031"],"award-info":[{"award-number":["2022C02031"]}]},{"name":"Agricultural Science and Technology Cooperation Program of Zhejiang Province","award":["LGF21C080001"],"award-info":[{"award-number":["LGF21C080001"]}]},{"name":"Agricultural Science and Technology Cooperation Program of Zhejiang Province","award":["LGC21C180001"],"award-info":[{"award-number":["LGC21C180001"]}]},{"name":"Key Research and Development Program of Zhejiang Province","award":["2022SNJF060"],"award-info":[{"award-number":["2022SNJF060"]}]},{"name":"Key Research and Development Program of Zhejiang Province","award":["2022C02031"],"award-info":[{"award-number":["2022C02031"]}]},{"name":"Key Research and Development Program of Zhejiang Province","award":["LGF21C080001"],"award-info":[{"award-number":["LGF21C080001"]}]},{"name":"Key Research and Development Program of Zhejiang Province","award":["LGC21C180001"],"award-info":[{"award-number":["LGC21C180001"]}]},{"name":"Zhejiang Province Public Welfare Technology Application research Project (CN)","award":["2022SNJF060"],"award-info":[{"award-number":["2022SNJF060"]}]},{"name":"Zhejiang Province Public Welfare Technology Application research Project (CN)","award":["2022C02031"],"award-info":[{"award-number":["2022C02031"]}]},{"name":"Zhejiang Province Public Welfare Technology Application research Project (CN)","award":["LGF21C080001"],"award-info":[{"award-number":["LGF21C080001"]}]},{"name":"Zhejiang Province Public Welfare Technology Application research Project (CN)","award":["LGC21C180001"],"award-info":[{"award-number":["LGC21C180001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Mycoplasma hyopneumoniae (Mhp), the primary pathogen causing Mycoplasma pneumonia of swine (MPS), brings massive economic losses worldwide. Genomic variability and post-translational protein modification can enhance the immune evasion of Mhp, which makes MPS prone to recurrent outbreaks on farms, even with vaccination or other treatments. The reverse vaccinology pipeline has been developed as an attractive potential method for vaccine development due to its high efficiency and applicability. In this study, a multi-epitope vaccine for Mhp was developed, and its immune responses were evaluated in mice and piglets. Genomic core proteins of Mhp were retrieved through pan-genome analysis, and four immunodominant antigens were screened by host homologous protein removal, membrane protein screening, and virulence factor identification. One immunodominant antigen, AAV27984.1 (membrane nuclease), was expressed by E. coli and named rMhp597. For epitope prioritization, 35 B-cell-derived epitopes were identified from the four immunodominant antigens, and 10 MHC-I and 6 MHC-II binding epitopes were further identified. The MHC-I\/II binding epitopes were merged and combined to produce recombinant proteins MhpMEV and MhpMEVC6His, which were used for animal immunization and structural analysis, respectively. Immunization of mice and piglets demonstrated that MhpMEV could induce humoral and cellular immune responses. The mouse serum antibodies could detect all 11 synthetic epitopes, and the piglet antiserum suppressed the nuclease activity of rMhp597. Moreover, piglet serum antibodies could also detect cultured Mhp strain 168. In summary, this study provides immunoassay results for a multi-epitope vaccine derived from the reverse vaccinology pipeline, and offers an alternative vaccine for MPS.<\/jats:p>","DOI":"10.3390\/ijms23147899","type":"journal-article","created":{"date-parts":[[2022,7,18]],"date-time":"2022-07-18T03:41:33Z","timestamp":1658115693000},"page":"7899","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Development of a Multi-Epitope Vaccine for Mycoplasma hyopneumoniae and Evaluation of Its Immune Responses in Mice and Piglets"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2433-0536","authenticated-orcid":false,"given":"Gaojian","family":"Li","sequence":"first","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4464-0398","authenticated-orcid":false,"given":"Jinqi","family":"Shu","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1750-1752","authenticated-orcid":false,"given":"Jing","family":"Jin","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1080-9108","authenticated-orcid":false,"given":"Jianhong","family":"Shu","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"},{"name":"Shaoxing Biomedical Research Institute, Zhejiang Sci-Tech University, Shaoxing 312000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4889-7609","authenticated-orcid":false,"given":"Huapeng","family":"Feng","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2735-3925","authenticated-orcid":false,"given":"Jian","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0265-5946","authenticated-orcid":false,"given":"Yulong","family":"He","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China"},{"name":"Shaoxing Biomedical Research Institute, Zhejiang Sci-Tech University, Shaoxing 312000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1016\/0378-1135(93)90035-6","article-title":"The nature and detection of mycoplasmal immunogens","volume":"37","author":"Ross","year":"1993","journal-title":"Vet. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1016\/j.vetmic.2007.09.008","article-title":"Control of Mycoplasma hyopneumoniae infections in pigs","volume":"126","author":"Maes","year":"2008","journal-title":"Vet. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1111\/tbed.12677","article-title":"Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control","volume":"65","author":"Maes","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/j.rvsc.2019.01.007","article-title":"A concise review of vaccines against Mycoplasma hyopneumoniae","volume":"123","author":"Tao","year":"2019","journal-title":"Res. Vet. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"510943","DOI":"10.3389\/fimmu.2020.510943","article-title":"Genomic Variability and Post-translational Protein Processing Enhance the Immune Evasion of Mycoplasma hyopneumoniae and Its Interaction with the Porcine Immune System","volume":"11","author":"Li","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.vetmic.2018.07.004","article-title":"Genotyping Mycoplasma hyopneumoniae isolates based on multi-locus sequence typing, multiple-locus variable-number tandem repeat analysis and analysing gene p146","volume":"222","author":"Felde","year":"2018","journal-title":"Vet. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2020","DOI":"10.1128\/JCM.00125-11","article-title":"Multiple-locus variable-number tandem-repeat analysis is a suitable tool for differentiation of Mycoplasma hyopneumoniae strains without cultivation","volume":"49","author":"Vranckx","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1840","DOI":"10.1111\/tbed.13233","article-title":"Mycoplasma hyopneumoniae variability: Current trends and proposed terminology for genomic classification","volume":"66","author":"Betlach","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.vetmic.2016.09.022","article-title":"Selective medium for culture of Mycoplasma hyopneumoniae","volume":"195","author":"Cook","year":"2016","journal-title":"Vet. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1080\/08830185.2019.1657426","article-title":"Immunoinformatics: In Silico Approaches and Computational Design of a Multi-epitope, Immunogenic Protein","volume":"38","author":"Bahrami","year":"2019","journal-title":"Int. Rev. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.1038\/s41598-018-19456-1","article-title":"Novel Immunoinformatics Approaches to Design Multi-epitope Subunit Vaccine for Malaria by Investigating Anopheles Salivary Protein","volume":"8","author":"Pandey","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1080\/21645515.2015.1012017","article-title":"In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations","volume":"11","author":"Yang","year":"2015","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5240","DOI":"10.1038\/s41598-019-41496-4","article-title":"Prioritization of potential vaccine targets using comparative proteomics and designing of the chimeric multi-epitope vaccine against Pseudomonas aeruginosa","volume":"9","author":"Solanki","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4409","DOI":"10.1038\/s41598-019-40833-x","article-title":"In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases","volume":"9","author":"Shey","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"9232","DOI":"10.1038\/s41598-017-09199-w","article-title":"Exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection","volume":"7","author":"Ali","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Wang, D., Mai, J., Zhou, W., Yu, W., Zhan, Y., Wang, N., Epstein, N.D., and Yang, Y. (2020). Immunoinformatic Analysis of T- and B-Cell Epitopes for SARS-CoV-2 Vaccine Design. Vaccines, 8.","DOI":"10.3390\/vaccines8030355"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1038\/s41586-021-03828-1","article-title":"Highly accurate protein structure prediction for the human proteome","volume":"596","author":"Tunyasuvunakool","year":"2021","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"669812","DOI":"10.3389\/fimmu.2021.669812","article-title":"Development of a Novel Multi-Epitope Vaccine Against Crimean-Congo Hemorrhagic Fever Virus: An Integrated Reverse Vaccinology, Vaccine Informatics and Biophysics Approach","volume":"12","author":"Ismail","year":"2021","journal-title":"Front. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"10097","DOI":"10.1074\/jbc.M110.208140","article-title":"Mhp107 is a member of the multifunctional adhesin family of Mycoplasma hyopneumoniae","volume":"286","author":"Seymour","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"33971","DOI":"10.1074\/jbc.M110.104463","article-title":"A processed multidomain Mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia","volume":"285","author":"Seymour","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1924","DOI":"10.1021\/pr201115v","article-title":"Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs","volume":"11","author":"Deutscher","year":"2012","journal-title":"J. Proteome Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.vetmic.2019.05.011","article-title":"Mycoplasma hyopneumoniae Mhp597 is a cytotoxicity, inflammation and immunosuppression associated nuclease","volume":"235","author":"Li","year":"2019","journal-title":"Vet. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1186\/s12929-021-00764-0","article-title":"Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity","volume":"28","author":"Lin","year":"2021","journal-title":"J. Biomed. Sci."},{"key":"ref_24","first-page":"167280","article-title":"The mechanism and consequences of SARS-CoV-2 spike-mediated fusion and syncytia formation","volume":"434","author":"Bernier","year":"2021","journal-title":"J. Mol. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e00439-21","DOI":"10.1128\/JB.00439-21","article-title":"Pyruvate oxidase as a key determinant of pneumococcal viability during transcytosis across brain endothelium","volume":"203","author":"Anil","year":"2021","journal-title":"J. Bacteriol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Sharma, N., Wang, C., Kessler, P., and Sen, G.C. (2021). Herpes simplex virus 1 evades cellular antiviral response by inducing microRNA-24, which attenuates STING synthesis. PLoS Pathog., 17.","DOI":"10.1371\/journal.ppat.1009950"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Dar, H.A., Zaheer, T., Shehroz, M., Ullah, N., Naz, K., Muhammad, S.A., Zhang, T., and Ali, A. (2019). Immunoinformatics-Aided Design and Evaluation of a Potential Multi-Epitope Vaccine against Klebsiella Pneumoniae. Vaccines, 7.","DOI":"10.3390\/vaccines7030088"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"104388","DOI":"10.1016\/j.meegid.2020.104388","article-title":"In silico designing of peptide based vaccine for Hepatitis viruses using reverse vaccinology approach","volume":"84","author":"Chaudhuri","year":"2020","journal-title":"Infect. Genet. Evol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2517","DOI":"10.1038\/s41598-019-39299-8","article-title":"Designing a multi-epitope based vaccine to combat Kaposi Sarcoma utilizing immunoinformatics approach","volume":"9","author":"Chauhan","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3011","DOI":"10.1007\/s00253-020-10420-6","article-title":"Protective immunity following vaccination with a recombinant multiple-epitope protein of bovine herpesvirus type I in a rabbit model","volume":"104","author":"Wen","year":"2020","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1186\/s12917-020-02560-8","article-title":"Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2","volume":"16","author":"Tao","year":"2020","journal-title":"BMC Vet. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2666","DOI":"10.1016\/S0264-410X(00)00498-9","article-title":"Adjuvants designed for veterinary and human vaccines","volume":"19","author":"Aucouturier","year":"2001","journal-title":"Vaccine"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/S1074-7613(00)80413-6","article-title":"IL-12-deficient mice are defective in IFN gamma production and type 1 cytokine responses","volume":"4","author":"Magram","year":"1996","journal-title":"Immunity"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1504","DOI":"10.4049\/jimmunol.156.4.1504","article-title":"Regulation of mucosal and systemic antibody responses by T helper cell subsets, macrophages, and derived cytokines following oral immunization with live recombinant Salmonella","volume":"156","author":"VanCott","year":"1996","journal-title":"J. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1186\/s13567-021-00941-x","article-title":"Perspectives for improvement of Mycoplasma hyopneumoniae vaccines in pigs","volume":"52","author":"Maes","year":"2021","journal-title":"Vet. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1186\/s13567-019-0709-0","article-title":"Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae","volume":"50","author":"Matthijs","year":"2019","journal-title":"Vet. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1093\/molbev\/msz284","article-title":"Estimating Pangenomes with Roary","volume":"37","author":"Sitto","year":"2020","journal-title":"Mol. Biol. Evol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3691","DOI":"10.1093\/bioinformatics\/btv421","article-title":"Roary: Rapid large-scale prokaryote pan genome analysis","volume":"31","author":"Page","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1093\/bioinformatics\/btq249","article-title":"PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes","volume":"26","author":"Yu","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Yu, C.S., Cheng, C.W., Su, W.C., Chang, K.C., Huang, S.W., Hwang, J.K., and Lu, C.H. (2014). CELLO2GO: A web server for protein subCELlular LOcalization prediction with functional gene ontology annotation. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0099368"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"D687","DOI":"10.1093\/nar\/gky1080","article-title":"VFDB 2019: A comparative pathogenomic platform with an interactive web interface","volume":"47","author":"Liu","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_42","first-page":"531","article-title":"Protein identification and analysis tools in the ExPASy server","volume":"112","author":"Wilkins","year":"1999","journal-title":"Methods Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1006\/jmbi.2000.4315","article-title":"Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes","volume":"305","author":"Krogh","year":"2001","journal-title":"J. Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1038\/s41587-019-0036-z","article-title":"SignalP 5.0 improves signal peptide predictions using deep neural networks","volume":"37","author":"Tsirigos","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1016\/0014-5793(90)80535-Q","article-title":"A semi-empirical method for prediction of antigenic determinants on protein antigens","volume":"276","author":"Kolaskar","year":"1990","journal-title":"FEBS Lett."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"W72","DOI":"10.1093\/nar\/gki396","article-title":"SCRATCH: A protein structure and structural feature prediction server","volume":"33","author":"Cheng","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/1471-2105-8-4","article-title":"VaxiJen: A server for prediction of protective antigens, tumour antigens and subunit vaccines","volume":"8","author":"Doytchinova","year":"2007","journal-title":"BMC Bioinform."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"W24","DOI":"10.1093\/nar\/gkx346","article-title":"BepiPred-2.0: Improving sequence-based B-cell epitope prediction using conformational epitopes","volume":"45","author":"Jespersen","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_49","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"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Saha, S., and Raghava, G.P.S. (2004, January 13\u201316). BcePred: Prediction of Continuous B-Cell Epitopes in Antigenic Sequences Using Physico-chemical Properties. Proceedings of the Artificial Immune Systems, Catania, Sicily, Italy.","DOI":"10.1007\/978-3-540-30220-9_16"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"2066","DOI":"10.1093\/bioinformatics\/btq324","article-title":"EpiTOP\u2014A proteochemometric tool for MHC class II binding prediction","volume":"26","author":"Dimitrov","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"3621","DOI":"10.1093\/nar\/gkg510","article-title":"MHCPred: A server for quantitative prediction of peptide-MHC binding","volume":"31","author":"Guan","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"278","DOI":"10.3389\/fimmu.2017.00278","article-title":"The Immune Epitope Database and Analysis Resource in Epitope Discovery and Synthetic Vaccine Design","volume":"8","author":"Fleri","year":"2017","journal-title":"Front. Immunol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1186\/1471-2105-9-62","article-title":"VirulentPred: A SVM based prediction method for virulent proteins in bacterial pathogens","volume":"9","author":"Garg","year":"2008","journal-title":"BMC Bioinform."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1186\/1745-6150-8-30","article-title":"Designing of interferon-gamma inducing MHC class-II binders","volume":"8","author":"Dhanda","year":"2013","journal-title":"Biol. Direct"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1093\/bioinformatics\/btv639","article-title":"Gapped sequence alignment using artificial neural networks: Application to the MHC class I system","volume":"32","author":"Andreatta","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Calis, J.J., Maybeno, M., Greenbaum, J.A., Weiskopf, D., De Silva, A.D., Sette, A., Kesmir, C., and Peters, B. (2013). Properties of MHC class I presented peptides that enhance immunogenicity. PLoS Comput. Biol., 9.","DOI":"10.1371\/journal.pcbi.1003266"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1186\/1746-6148-10-43","article-title":"A recombinant chimera comprising the R1 and R2 repeat regions of M. hyopneumoniae P97 and the N-terminal region of A. pleuropneumoniae ApxIII elicits immune responses","volume":"10","author":"Lee","year":"2014","journal-title":"BMC Vet. Res."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"W430","DOI":"10.1093\/nar\/gkw306","article-title":"RaptorX-Property: A web server for protein structure property prediction","volume":"44","author":"Wang","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"e136","DOI":"10.1017\/S0950268821001229","article-title":"Design of a new multi-epitope vaccine against Brucella based on T and B cell epitopes using bioinformatics methods","volume":"149","author":"Chen","year":"2021","journal-title":"Epidemiol. Infect."},{"key":"ref_61","doi-asserted-by":"crossref","unstructured":"Ashfaq, U.A., Saleem, S., Masoud, M.S., Ahmad, M., Nahid, N., Bhatti, R., Almatroudi, A., and Khurshid, M. (2021). Rational design of multi epitope-based subunit vaccine by exploring MERS-COV proteome: Reverse vaccinology and molecular docking approach. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0245072"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"107265","DOI":"10.1016\/j.intimp.2020.107265","article-title":"Immuno-informatics analysis and expression of a novel multi-domain antigen as a vaccine candidate against glioblastoma","volume":"91","author":"Gharbavi","year":"2021","journal-title":"Int. Immunopharmacol."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"W407","DOI":"10.1093\/nar\/gkm290","article-title":"ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins","volume":"35","author":"Wiederstein","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: A program to check the stereochemical quality of protein structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Crystallogr."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"1771","DOI":"10.1093\/bioinformatics\/btu097","article-title":"ZDOCK server: Interactive docking prediction of protein-protein complexes and symmetric multimers","volume":"30","author":"Pierce","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"776","DOI":"10.2174\/0929866526666190430115052","article-title":"Immunoenhancement of Recombinant Neisseria meningitides PorB Protein on Porcine Circovirus Type 2 and Mycoplasma hyopneumoniae Genetically Engineered Vaccines","volume":"26","author":"Yang","year":"2019","journal-title":"Protein Pept. Lett."},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Tao, Y., Li, G., Zheng, W., Shu, J., Chen, J., Yang, F., Wu, Y., and He, Y. (2019). Development of a Combined Genetic Engineering Vaccine for Porcine Circovirus Type 2 and Mycoplasma hyopneumoniae by a Baculovirus Expression System. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20184425"},{"key":"ref_68","first-page":"199","article-title":"Ethical Considerations in Animal Research: The Principle of 3R\u2019s","volume":"73","author":"Zambrano","year":"2020","journal-title":"Rev. Investig. Clin. Organo Hosp. Enferm. Nutr."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/1479-5876-4-42","article-title":"Immunological consequences of using three different clinical\/laboratory techniques of emulsifying peptide-based vaccines in incomplete Freund\u2019s adjuvant","volume":"4","author":"Koh","year":"2006","journal-title":"J. Transl. Med."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1016\/S0264-410X(96)00055-2","article-title":"International bank for foot-and-mouth disease vaccine: Assessment of Montanide ISA 25 and ISA 206, two commercially available oil adjuvants","volume":"14","author":"Barnett","year":"1996","journal-title":"Vaccine"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"S44","DOI":"10.1016\/j.vaccine.2005.01.116","article-title":"The use of oil adjuvants in therapeutic vaccines","volume":"24","author":"Aucouturier","year":"2006","journal-title":"Vaccine"},{"key":"ref_72","doi-asserted-by":"crossref","unstructured":"Chang, A.M., Chen, C.C., Hou, D.L., Ke, G.M., and Lee, J.W. (2021). Effects of a Recombinant Gonadotropin-Releasing Hormone Vaccine on Reproductive Function in Adult Male ICR Mice. Vaccines, 9.","DOI":"10.3390\/vaccines9080808"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1007\/s10529-020-02792-6","article-title":"Development of HPV(16,18,31,45) E5 and E7 peptides-based vaccines predicted by immunoinformatics tools","volume":"42","author":"Namvar","year":"2020","journal-title":"Biotechnol. Lett."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1016\/j.jviromet.2011.11.023","article-title":"Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: Baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2","volume":"179","author":"Xu","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"7784","DOI":"10.1128\/IAI.73.11.7784-7787.2005","article-title":"In vivo expression analysis of the P97 and P102 paralog families of Mycoplasma hyopneumoniae","volume":"73","author":"Adams","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"e00282-11","DOI":"10.1128\/mBio.00282-11","article-title":"Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography","volume":"3","author":"Bogema","year":"2012","journal-title":"mBio"},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1128\/CDLI.7.4.528-535.2000","article-title":"Species-specific monoclonal antibodies to Escherichia coli-expressed p36 cytosolic protein of Mycoplasma hyopneumoniae","volume":"7","author":"Caron","year":"2000","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"5734","DOI":"10.1016\/j.vaccine.2006.04.036","article-title":"A recombinant chimera composed of R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile enterotoxin B subunit elicits immune response in mice","volume":"24","author":"Conceicao","year":"2006","journal-title":"Vaccine"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1016\/j.vetmic.2017.01.023","article-title":"A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice","volume":"201","author":"Jorge","year":"2017","journal-title":"Vet. Microbiol."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.vaccine.2012.10.088","article-title":"Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines","volume":"31","author":"Galli","year":"2012","journal-title":"Vaccine"},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1107\/S2059798320003903","article-title":"Structure of P46, an immunodominant surface protein from Mycoplasma hyopneumoniae: Interaction with a monoclonal antibody","volume":"76","author":"Guasch","year":"2020","journal-title":"Acta Crystallogr. D Struct. Biol."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"2947","DOI":"10.1093\/bioinformatics\/btm404","article-title":"Clustal W and Clustal X version 2.0","volume":"23","author":"Larkin","year":"2007","journal-title":"Bioinformatics"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"W293","DOI":"10.1093\/nar\/gkab301","article-title":"Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation","volume":"49","author":"Letunic","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1016\/j.vetmic.2009.10.007","article-title":"Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array","volume":"142","author":"Meens","year":"2010","journal-title":"Vet. Microbiol."},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"3056","DOI":"10.1128\/IAI.68.5.3056-3060.2000","article-title":"R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia","volume":"68","author":"Minion","year":"2000","journal-title":"Infect. Immun."},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"5891","DOI":"10.1021\/pr400903s","article-title":"P159 from Mycoplasma hyopneumoniae binds porcine cilia and heparin and is cleaved in a manner akin to ectodomain shedding","volume":"12","author":"Raymond","year":"2013","journal-title":"J. Proteome Res."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"5790","DOI":"10.1128\/JB.186.17.5790-5798.2004","article-title":"Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids","volume":"186","author":"Schmidt","year":"2004","journal-title":"J. Bacteriol."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"2920","DOI":"10.1021\/pr500087c","article-title":"Cilium adhesin P216 (MHJ_0493) is a target of ectodomain shedding and aminopeptidase activity on the surface of Mycoplasma hyopneumoniae","volume":"13","author":"Tacchi","year":"2014","journal-title":"J. Proteome Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/7899\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:52:41Z","timestamp":1760140361000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/7899"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,18]]},"references-count":88,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["ijms23147899"],"URL":"https:\/\/doi.org\/10.3390\/ijms23147899","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,7,18]]}}}