{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T22:31:37Z","timestamp":1792189897457,"version":"4.4.0"},"reference-count":81,"publisher":"Public Library of Science (PLoS)","issue":"11","license":[{"start":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T00:00:00Z","timestamp":1762387200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"the Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82360394"],"award-info":[{"award-number":["82360394"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"the Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82460399"],"award-info":[{"award-number":["82460399"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"the Youth Science and Technology Top Talent Project","award":["2022TSYCCX0112"],"award-info":[{"award-number":["2022TSYCCX0112"]}]},{"name":"the Outstanding Youth Project of the Autonomous Region","award":["2023D01E12"],"award-info":[{"award-number":["2023D01E12"]}]},{"name":"the Central Government Guides Local Science and Technology Development Fund Project","award":["ZYYD2025ZY19"],"award-info":[{"award-number":["ZYYD2025ZY19"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Brucellosis, caused by the intracellular pathogen\n                    <jats:italic>\n                      <jats:italic>Brucella<\/jats:italic>\n                    <\/jats:italic>\n                    , remains a significant health challenge, alongside substantial economic impacts on livestock industries. Despite antibiotic treatments, the absence of licensed human vaccines necessitates innovative preventive strategies. In this study, we employed reverse vaccinology to design a novel multi-epitope vaccine (MEV) targeting\n                    <jats:italic>\n                      <jats:italic>Brucella melitensis<\/jats:italic>\n                    <\/jats:italic>\n                    . Three immunogenic proteins\u2014outer membrane protein OMP31, LPS assembly protein LptE, and the type IV secretion system protein VirB2\u2014were selected as vaccine candidates. Comprehensive bioinformatics analysis identified six cytotoxic T lymphocyte (CTL) epitopes, nine helper T lymphocyte (HTL) epitopes, seven linear B-cell epitopes, and five conformational B-cell epitopes. The incorporation of molecular adjuvants (cholera toxin B subunit and PADRE) served to further enhance the immunogenicity of the vaccine. Given that Brucella is an intracellular parasite, TAT cell-penetrating peptides were added to further enhance the intracellular delivery of MEV. The constructed MEV has been shown to have excellent antigenicity (VaxiJen score &gt;0.8), stability (instability index &lt;40), solubility (Protein-Sol score: 0.87) and hydrophilicity (GRAVY index: \u22120.319), and is non-allergenic. Structural optimization, including disulfide bond engineering (11 pairs of residues), improved molecular stability, with molecular docking and dynamics simulations confirming robust interactions with immune cell receptors (docking score: \u2212311.85). Using SnapGene 7.1.2, we performed\n                    <jats:italic>\n                      <jats:italic>in silico<\/jats:italic>\n                    <\/jats:italic>\n                    cloning simulation of the codon-optimized multi-epitope vaccine (MEV) sequence into the pMV261 shuttle vector, generating a recombinant BCG (rBCG) construct. Immunoinformatics simulations (C-ImmSim) demonstrated potent immune activation, with significant increases in cytotoxic T cells (1050 cells\/mm\u00b3), memory helper T cells (1150 cells\/mm\u00b3), and IFN-\u03b3 production (2\u2009\u00d7\u200910^6\u2009ng\/ml), alongside sustained IgG\/IgM titers over 350 days(1\u2009\u00d7\u200910^5 cells\/mm\n                    <jats:sup>3<\/jats:sup>\n                    ) . Furthermore, the recombinant BCG multi-epitope Brucella vaccine, developed through bioinformatics approaches, demonstrates promising characteristics and immunogenicity. Nevertheless, its immunological efficacy requires to further experimental validation.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0334843","type":"journal-article","created":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T18:40:47Z","timestamp":1762454447000},"page":"e0334843","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":10,"title":["Design of a multi-epitope recombinant BCG vaccine targeting Brucella OMP31, LptE and VirB2 in immunoinformatics approaches"],"prefix":"10.1371","volume":"20","author":[{"given":"Chuang","family":"Li","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuejie","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingxing","family":"Qi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenglong","family":"Chai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiarui","family":"Luo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaiyu","family":"Shang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingting","family":"Tian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huidong","family":"Shi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingzhe","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruixue","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuling","family":"Pu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junyu","family":"Kuang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5795-8672","authenticated-orcid":true,"given":"Fengbo","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,11,6]]},"reference":[{"key":"pone.0334843.ref001","article-title":"Pathophysiology, current therapeutic options, vaccine candidates, and drug targets for human brucellosis","volume":"17","author":"M Pritam","year":"2024","journal-title":"Curr Mol Pharmacol"},{"key":"pone.0334843.ref002","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1146\/annurev-micro-090110-102905","article-title":"Interactions of the human pathogenic Brucella species with their hosts","volume":"65","author":"VL Atluri","year":"2011","journal-title":"Annu Rev Microbiol"},{"issue":"12","key":"pone.0334843.ref003","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pmed.0040317","article-title":"Perspectives for the treatment of brucellosis in the 21st century: the Ioannina recommendations","volume":"4","author":"J Ariza","year":"2007","journal-title":"PLoS Med"},{"issue":"1","key":"pone.0334843.ref004","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1186\/s40249-020-00715-1","article-title":"Human brucellosis: recent advances and future challenges","volume":"9","author":"D O\u2019Callaghan","year":"2020","journal-title":"Infect Dis Poverty"},{"issue":"10","key":"pone.0334843.ref005","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.2174\/1386207325666220930124013","article-title":"Computer aided reverse vaccinology: a game-changer approach for vaccine development","volume":"26","author":"P Srivastava","year":"2023","journal-title":"Comb Chem High Throughput Screen"},{"issue":"2","key":"pone.0334843.ref006","doi-asserted-by":"crossref","first-page":"2295398","DOI":"10.1080\/07853890.2023.2295398","article-title":"Brucellosis: epidemiology, pathogenesis, diagnosis and treatment-a comprehensive review","volume":"55","author":"KA Qureshi","year":"2023","journal-title":"Ann Med"},{"issue":"1726","key":"pone.0334843.ref007","doi-asserted-by":"crossref","first-page":"20160224","DOI":"10.1098\/rstb.2016.0224","article-title":"Insertion of proteins and lipopolysaccharide into the bacterial outer membrane","volume":"372","author":"I Botos","year":"2017","journal-title":"Philos Trans R Soc Lond B Biol Sci"},{"issue":"24","key":"pone.0334843.ref008","doi-asserted-by":"crossref","first-page":"5169","DOI":"10.1016\/j.vaccine.2006.04.005","article-title":"Evaluation of novel Brucella melitensis unmarked deletion mutants for safety and efficacy in the goat model of brucellosis","volume":"24","author":"MM Kahl-McDonagh","year":"2006","journal-title":"Vaccine"},{"key":"pone.0334843.ref009","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.sbi.2014.02.006","article-title":"Recent advances in the structural and molecular biology of type IV secretion systems","volume":"27","author":"M Trokter","year":"2014","journal-title":"Curr Opin Struct Biol"},{"issue":"2","key":"pone.0334843.ref010","doi-asserted-by":"crossref","first-page":"1155","DOI":"10.1007\/s10989-019-09918-z","article-title":"Epitope prediction by novel immunoinformatics approach: a state-of-the-art review","volume":"26","author":"E Raoufi","year":"2020","journal-title":"Int J Pept Res Ther"},{"issue":"6","key":"pone.0334843.ref011","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1038\/s41573-021-00163-y","article-title":"Emerging concepts in the science of vaccine adjuvants","volume":"20","author":"B Pulendran","year":"2021","journal-title":"Nat Rev Drug Discov"},{"issue":"5","key":"pone.0334843.ref012","doi-asserted-by":"crossref","first-page":"802","DOI":"10.3390\/vaccines10050802","article-title":"Auxotrophic mycobacterium bovis bcg: updates and perspectives","volume":"10","author":"OA Dellagostin","year":"2022","journal-title":"Vaccines (Basel)"},{"key":"pone.0334843.ref013","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1038\/s41541-020-00245-9","article-title":"Vaccine strategies for the Mtb\/HIV copandemic","volume":"5","author":"R Sharan","year":"2020","journal-title":"NPJ Vaccines"},{"key":"pone.0334843.ref014","doi-asserted-by":"crossref","first-page":"664212","DOI":"10.3389\/fimmu.2021.664212","article-title":"A recombinant BCG vaccine is safe and immunogenic in neonatal calves and reduces the clinical disease caused by the respiratory syncytial virus","volume":"12","author":"FE D\u00edaz","year":"2021","journal-title":"Front Immunol"},{"key":"pone.0334843.ref015","first-page":"531","article-title":"Protein identification and analysis tools in the ExPASy server","volume":"112","author":"MR Wilkins","year":"1999","journal-title":"Methods Mol Biol"},{"key":"pone.0334843.ref016","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":"IA Doytchinova","year":"2007","journal-title":"BMC Bioinformatics"},{"issue":"5","key":"pone.0334843.ref017","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1016\/j.vaccine.2006.09.032","article-title":"Identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties","volume":"25","author":"IA Doytchinova","year":"2007","journal-title":"Vaccine"},{"issue":"6","key":"pone.0334843.ref018","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1093\/bioinformatics\/btt619","article-title":"AllergenFP: allergenicity prediction by descriptor fingerprints","volume":"30","author":"I Dimitrov","year":"2014","journal-title":"Bioinformatics"},{"key":"pone.0334843.ref019","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega","volume":"7","author":"F Sievers","year":"2011","journal-title":"Mol Syst Biol"},{"issue":"7","key":"pone.0334843.ref020","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1038\/s41587-021-01156-3","article-title":"SignalP 6.0 predicts all five types of signal peptides using protein language models","volume":"40","author":"F Teufel","year":"2022","journal-title":"Nat Biotechnol"},{"issue":"8","key":"pone.0334843.ref021","doi-asserted-by":"crossref","first-page":"1652","DOI":"10.1110\/ps.0303703","article-title":"Prediction of lipoprotein signal peptides in Gram-negative bacteria","volume":"12","author":"AS Juncker","year":"2003","journal-title":"Protein Sci"},{"issue":"11","key":"pone.0334843.ref022","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0013458","article-title":"Allele polymorphism and haplotype diversity of HLA-A, -B and -DRB1 loci in sequence-based typing for Chinese Uyghur ethnic group","volume":"5","author":"C Shen","year":"2010","journal-title":"PLoS One"},{"issue":"14","key":"pone.0334843.ref023","doi-asserted-by":"crossref","DOI":"10.1016\/j.heliyon.2024.e34721","article-title":"Development of a novel multi-epitope vaccine for brucellosis prevention","volume":"10","author":"K Shang","year":"2024","journal-title":"Heliyon"},{"key":"pone.0334843.ref024","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gkaa379","article-title":"NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data","volume":"48","author":"B Reynisson","year":"2020","journal-title":"Nucleic Acids Res"},{"issue":"6","key":"pone.0334843.ref025","doi-asserted-by":"crossref","first-page":"2304","DOI":"10.1021\/acs.jproteome.9b00874","article-title":"Improved prediction of MHC II antigen presentation through integration and motif deconvolution of mass spectrometry MHC eluted ligand data","volume":"19","author":"B Reynisson","year":"2020","journal-title":"J Proteome Res"},{"key":"pone.0334843.ref026","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gkx346","article-title":"BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes","volume":"45","author":"MC Jespersen","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"pone.0334843.ref027","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1186\/1471-2105-9-514","article-title":"ElliPro: a new structure-based tool for the prediction of antibody epitopes","volume":"9","author":"J Ponomarenko","year":"2008","journal-title":"BMC Bioinform"},{"issue":"9","key":"pone.0334843.ref028","doi-asserted-by":"crossref","first-page":"1408","DOI":"10.3390\/v16091408","article-title":"Targeting yezo virus structural proteins for multi-epitope vaccine design using immunoinformatics approach","volume":"16","author":"S Rahman","year":"2024","journal-title":"Viruses"},{"issue":"5","key":"pone.0334843.ref029","doi-asserted-by":"crossref","first-page":"1829","DOI":"10.1038\/s41596-020-0312-x","article-title":"The HDOCK server for integrated protein-protein docking","volume":"15","author":"Y Yan","year":"2020","journal-title":"Nat Protoc"},{"key":"pone.0334843.ref030","article-title":"HDOCK: a web server for protein-protein and protein-DNA\/RNA docking based on a hybrid strategy","volume":"45","author":"Y Yan","year":"2017","journal-title":"Nucleic Acids Res"},{"issue":"3","key":"pone.0334843.ref031","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1002\/prot.25234","article-title":"Addressing recent docking challenges: A hybrid strategy to integrate template-based and free protein-protein docking","volume":"85","author":"Y Yan","year":"2017","journal-title":"Proteins"},{"issue":"8","key":"pone.0334843.ref032","article-title":"Design of multi-epitope vaccine candidate against Brucella type IV secretion system (T4SS)","volume":"18","author":"Z Yin","year":"2023","journal-title":"PLoS One"},{"key":"pone.0334843.ref033","doi-asserted-by":"crossref","first-page":"111351","DOI":"10.1016\/j.intimp.2023.111351","article-title":"A multi-epitope subunit vaccine based on CU\/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB\/C mice","volume":"127","author":"Y Wang","year":"2024","journal-title":"Int Immunopharmacol"},{"issue":"9","key":"pone.0334843.ref034","article-title":"Development of innovative multi-epitope mRNA vaccine against central nervous system tuberculosis using in silico approaches","volume":"19","author":"H Shi","year":"2024","journal-title":"PLoS One"},{"key":"pone.0334843.ref035","first-page":"101745","article-title":"Development of an ancestral DC and TLR4-inducing multi-epitope peptide vaccine against the spike protein of SARS-CoV and SARS-CoV-2 using the advanced immunoinformatics approaches","volume":"39","author":"C Aram","year":"2024","journal-title":"Biochem Biophys Rep"},{"key":"pone.0334843.ref036","doi-asserted-by":"crossref","first-page":"884433","DOI":"10.3389\/fimmu.2022.884433","article-title":"Design of a recombinant multivalent epitope vaccine based on SARS-CoV-2 and its variants in immunoinformatics approaches","volume":"13","author":"M Yu","year":"2022","journal-title":"Front Immunol"},{"issue":"1","key":"pone.0334843.ref037","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1186\/s43141-023-00614-6","article-title":"Immunoinformatics analysis of Brucella melitensis to approach a suitable vaccine against brucellosis","volume":"21","author":"P Hashemzadeh","year":"2023","journal-title":"J Genet Eng Biotechnol"},{"issue":"6","key":"pone.0334843.ref038","first-page":"681","article-title":"SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments","volume":"11","author":"C Geourjon","year":"1995","journal-title":"Comput Appl Biosci"},{"issue":"6557","key":"pone.0334843.ref039","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1126\/science.abj8754","article-title":"Accurate prediction of protein structures and interactions using a three-track neural network","volume":"373","author":"M Baek","year":"2021","journal-title":"Science"},{"key":"pone.0334843.ref040","article-title":"GalaxyRefine: protein structure refinement driven by side-chain repacking","volume":"41","author":"L Heo","year":"2013","journal-title":"Nucleic Acids Res"},{"key":"pone.0334843.ref041","first-page":"293","article-title":"Effective protein model structure refinement by loop modeling and overall relaxation","author":"GR Lee","year":"2016","journal-title":"Proteins"},{"issue":"3","key":"pone.0334843.ref042","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1006\/jmbi.1996.0663","article-title":"The NMR solution structure of intestinal fatty acid-binding protein complexed with palmitate: application of a novel distance geometry algorithm","volume":"264","author":"ME Hodsdon","year":"1996","journal-title":"J Mol Biol"},{"issue":"9","key":"pone.0334843.ref043","doi-asserted-by":"crossref","first-page":"1511","DOI":"10.1002\/pro.5560020916","article-title":"Verification of protein structures: patterns of nonbonded atomic interactions","volume":"2","author":"C Colovos","year":"1993","journal-title":"Protein Sci"},{"key":"pone.0334843.ref044","doi-asserted-by":"crossref","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":"M Wiederstein","year":"2007","journal-title":"Nucleic Acids Res"},{"key":"pone.0334843.ref045","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1186\/1471-2105-14-346","article-title":"Disulfide by design 2.0: a web-based tool for disulfide engineering in proteins","volume":"14","author":"DB Craig","year":"2013","journal-title":"BMC Bioinformatics"},{"issue":"1","key":"pone.0334843.ref046","doi-asserted-by":"crossref","first-page":"10330","DOI":"10.1038\/s41598-020-67230-z","article-title":"Prediction of disulfide bond engineering sites using a machine learning method","volume":"10","author":"X Gao","year":"2020","journal-title":"Sci Rep"},{"key":"pone.0334843.ref047","doi-asserted-by":"crossref","first-page":"812774","DOI":"10.3389\/fimmu.2022.812774","article-title":"Toll-like receptor signaling and its role in cell-mediated immunity","volume":"13","author":"T Duan","year":"2022","journal-title":"Front Immunol"},{"issue":"2","key":"pone.0334843.ref048","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.bjid.2015.10.011","article-title":"TLR2 and TLR4 mediated host immune responses in major infectious diseases: a review","volume":"20","author":"S Mukherjee","year":"2016","journal-title":"Braz J Infect Dis"},{"issue":"10","key":"pone.0334843.ref049","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.1021\/ci200227u","article-title":"LigPlot+: multiple ligand-protein interaction diagrams for drug discovery","volume":"51","author":"RA Laskowski","year":"2011","journal-title":"J Chem Inf Model"},{"issue":"5","key":"pone.0334843.ref050","article-title":"Designing a conjugate vaccine targeting Klebsiella pneumoniae ST258 and ST11","volume":"10","author":"M Li","year":"2024","journal-title":"Heliyon"},{"key":"pone.0334843.ref051","article-title":"iMODS: internal coordinates normal mode analysis server","volume":"42","author":"JR L\u00f3pez-Blanco","year":"2014","journal-title":"Nucleic Acids Res"},{"issue":"1","key":"pone.0334843.ref052","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1186\/s12859-023-05234-y","article-title":"Visual dynamics: a WEB application for molecular dynamics simulation using GROMACS","volume":"24","author":"IHP Vieira","year":"2023","journal-title":"BMC Bioinformatics"},{"key":"pone.0334843.ref053","doi-asserted-by":"crossref","first-page":"4849","DOI":"10.1016\/j.csbj.2023.09.024","article-title":"CHAPERONg: a tool for automated GROMACS-based molecular dynamics simulations and trajectory analyses","volume":"21","author":"AA Yekeen","year":"2023","journal-title":"Comput Struct Biotechnol J"},{"issue":"1","key":"pone.0334843.ref054","doi-asserted-by":"crossref","first-page":"96","DOI":"10.3934\/microbiol.2021007","article-title":"Bacillus Calmette-Guerin (BCG): the adroit vaccine","volume":"7","author":"OA Adesanya","year":"2021","journal-title":"AIMS Microbiol"},{"issue":"5","key":"pone.0334843.ref055","doi-asserted-by":"crossref","DOI":"10.1136\/jitc-2021-002694","article-title":"Identification and validation of viral antigens sharing sequence and structural homology with tumor-associated antigens (TAAs)","volume":"9","author":"C Ragone","year":"2021","journal-title":"J Immunother Cancer"},{"key":"pone.0334843.ref056","doi-asserted-by":"crossref","first-page":"998262","DOI":"10.3389\/fimmu.2022.998262","article-title":"In-silico evaluation of adenoviral COVID-19 vaccination protocols: assessment of immunological memory up to 6 months after the third dose","volume":"13","author":"P Stolfi","year":"2022","journal-title":"Front Immunol"},{"issue":"4","key":"pone.0334843.ref057","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0009862","article-title":"Computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system","volume":"5","author":"N Rapin","year":"2010","journal-title":"PLoS One"},{"issue":"3","key":"pone.0334843.ref058","doi-asserted-by":"crossref","first-page":"654","DOI":"10.3390\/vaccines11030654","article-title":"The development of diagnostic and vaccine strategies for early detection and control of human brucellosis, particularly in endemic areas","volume":"11","author":"A Elbehiry","year":"2023","journal-title":"Vaccines (Basel)"},{"key":"pone.0334843.ref059","doi-asserted-by":"crossref","first-page":"122986","DOI":"10.1016\/j.lfs.2024.122986","article-title":"The development of a human Brucella mucosal vaccine: What should be considered?","volume":"355","author":"T Tian","year":"2024","journal-title":"Life Sci"},{"key":"pone.0334843.ref060","doi-asserted-by":"crossref","first-page":"281","DOI":"10.4142\/jvs.2017.18.S1.281","article-title":"Development and trial of vaccines against Brucella","volume":"18","author":"J Lalsiamthara","year":"2017","journal-title":"J Vet Sci"},{"key":"pone.0334843.ref061","doi-asserted-by":"crossref","first-page":"105057","DOI":"10.1016\/j.compbiomed.2021.105057","article-title":"An overview of progress from empirical to rational design in modern vaccine development, with an emphasis on computational tools and immunoinformatics approaches","volume":"140","author":"S Soleymani","year":"2022","journal-title":"Comput Biol 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Coleman","year":"1985","journal-title":"Cell"},{"key":"pone.0334843.ref071","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/978-1-0716-3239-0_25","article-title":"Immunoinformatics protocol to design multi-epitope subunit vaccines","volume":"2673","author":"P Kalita","year":"2023","journal-title":"Methods Mol Biol"},{"issue":"9270","key":"pone.0334843.ref072","doi-asserted-by":"crossref","first-page":"1777","DOI":"10.1016\/S0140-6736(00)04904-7","article-title":"An overview of the immune system","volume":"357","author":"J Parkin","year":"2001","journal-title":"Lancet"},{"key":"pone.0334843.ref073","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1146\/annurev-biodatasci-021920-100259","article-title":"Immunoinformatics: predicting peptide-MHC binding","volume":"3","author":"M Nielsen","year":"2020","journal-title":"Annu Rev Biomed Data 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