{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T18:07:54Z","timestamp":1785607674205,"version":"3.56.0"},"reference-count":114,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2025,7,20]],"date-time":"2025-07-20T00:00:00Z","timestamp":1752969600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004704","name":"National Research Council of Thailand","doi-asserted-by":"publisher","award":["NRCT5-RGJ63010-115"],"award-info":[{"award-number":["NRCT5-RGJ63010-115"]}],"id":[{"id":"10.13039\/501100004704","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Fasciola gigantica (F. gigantica) is a vital parasite that causes fasciolosis. Liver fluke infections affect livestock animals, and the Fasciola species (Fasciola spp.) vaccine has been tested for many types of these diseases. Currently, computer-based vaccine design represents an attractive alternative for constructing vaccines. Thus, this study aimed to design the epitopes of linear B-cells (BCL) and helper T lymphocytes (HTL) using an immunoinformatic approach and to investigate in silico and the mice\u2019s immune response. A non-conserved host region, overlapping F. gigantica cathepsin B proteins (FgCatB), and the highest conserved residue percentages were the criteria used to construct epitopes. The GPGPG linker was used to link epitopes in the multi-epitope Fasciola gigantica cathepsin B (MeFgCatB) peptide. The MeFgCatB peptide has high antigenicity, non-allergenicity, non-toxicity, good solubility, and a high-quality structure. The molecular docking between the MeFgCatB peptide and Toll-like receptor 2 (TLR-2) was evaluated. The IgM, IgG1, and IgG2 levels were elevated in silico. In mice, the MeFgCatB peptide was synthesized and administered as an injection. The MeFgCatB-specific IgG1 and IgG2a levels were elevated after week 2, showing a predominance of IgG1. The rFgCatB1, rFgCatB2, and rFgCatB3 were detected using the MeFgCatB peptide-immunized sera. The MeFgCatB peptide-immunized sera were detected at approximately 28\u201334 kDa in the whole body. In addition, the MeFgCatB immunized sera can positively signal at the caecal epithelium in the NEJ, 4WKJ, and adult stages. In summary, the MeFgCatB peptide is able to induce mixed Th1\/Th2 immune responses with Th2 dominating and to detect the native protein of F. gigantica. The MeFgCatB peptide should help against F. gigantica in future experiments.<\/jats:p>","DOI":"10.3390\/ijms26146971","type":"journal-article","created":{"date-parts":[[2025,7,21]],"date-time":"2025-07-21T09:33:53Z","timestamp":1753090433000},"page":"6971","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Design of a Multi-Epitope Vaccine Based on Fasciola gigantica Cathepsin B and Evaluation of Immunological Responses in Mice"],"prefix":"10.3390","volume":"26","author":[{"given":"Supanan","family":"Chansap","sequence":"first","affiliation":[{"name":"Research Unit of Vaccine and Diagnosis of Parasitic Diseases, Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Road, Saen Sook Sub-District, Mueang District, Chonburi 20131, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Werachon","family":"Cheukamud","sequence":"additional","affiliation":[{"name":"Research Unit of Vaccine and Diagnosis of Parasitic Diseases, Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Road, Saen Sook Sub-District, Mueang District, Chonburi 20131, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thitikul","family":"Suthisintong","sequence":"additional","affiliation":[{"name":"Research Unit of Vaccine and Diagnosis of Parasitic Diseases, Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Road, Saen Sook Sub-District, Mueang District, Chonburi 20131, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pornanan","family":"Kueakhai","sequence":"additional","affiliation":[{"name":"Research Unit of Vaccine and Diagnosis of Parasitic Diseases, Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Road, Saen Sook Sub-District, Mueang District, Chonburi 20131, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Narin","family":"Changklungmoa","sequence":"additional","affiliation":[{"name":"Research Unit of Vaccine and Diagnosis of Parasitic Diseases, Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Road, Saen Sook Sub-District, Mueang District, Chonburi 20131, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2015\/786195","article-title":"Fascioliasis: An Ongoing Zoonotic Trematode Infection","volume":"1","author":"Nyindo","year":"2015","journal-title":"BioMed Res. Int."},{"key":"ref_2","first-page":"76","article-title":"Field Trials to Evaluate Five Fasciolicides against Natural Liver Fluke Infection in Cattle and Sheep in Egypt","volume":"12","author":"Mostafa","year":"2023","journal-title":"Int. J. Vet. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1309\/LMLFBB8PW4SA0YJI","article-title":"Fascioliasis Due to Fasciola hepatica and Fasciola gigantica Infection: An Update on This \u201cneglected\u201d Neglected Tropical Disease","volume":"42","author":"Tolan","year":"2011","journal-title":"Lab. Med."},{"key":"ref_4","first-page":"20499361231185413","article-title":"The Global Prevalence of Human Fascioliasis: A Systematic Review and Meta-Analysis","volume":"10","author":"Mayer","year":"2023","journal-title":"Ther. Adv. Infect. Dis."},{"key":"ref_5","first-page":"224","article-title":"Fibrosis and Collagen-I Accumulation in Bali Cattle Liver Tissue Infected with Fasciola gigantica","volume":"12","author":"Winaya","year":"2023","journal-title":"Int. J. Vet. Sci."},{"key":"ref_6","unstructured":"WHO (2007). Report of the WHO Informal Meeting on Use of Triclabendazole in Fascioliasis Control, WHO."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1079\/JOH2005298","article-title":"Triclabendazole: New Skills to Unravel an Old(Ish) Enigma","volume":"79","author":"Fairweather","year":"2005","journal-title":"J. Helminthol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"792","DOI":"10.1074\/mcp.RA117.000445","article-title":"Infection by the Helminth Parasite Fasciola Hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host","volume":"17","author":"Cwiklinski","year":"2018","journal-title":"Mol. Cell. Proteom."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Beesley, N.J., Cwiklinski, K., Allen, K., Hoyle, R.C., Spithill, T.W., James La Course, E., Williams, D.J.L., Paterson, S., and Hodgkinson, J.E. (2023). A Major Locus Confers Triclabendazole Resistance in Fasciola Hepatica and Shows Dominant Inheritance. PLoS Pathog., 19.","DOI":"10.1371\/journal.ppat.1011081"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/S0304-4017(96)01067-9","article-title":"Triclabendazole Treatment in Experimental Goat Fasciolosis: Anthelmintic Efficacy and Influence in Antibody Response and Pathophysiology of the Disease","volume":"68","author":"Becerra","year":"1997","journal-title":"Vet. Parasitol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1016\/j.pt.2016.03.002","article-title":"Current Threat of Triclabendazole Resistance in Fasciola Hepatica","volume":"32","author":"Kelley","year":"2016","journal-title":"Trends. Parasitol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Cabada, M.M., Lopez, M., Cruz, M., Delgado, J.R., Hill, V., and White, A.C. (2016). Treatment Failure after Multiple Courses of Triclabendazole among Patients with Fascioliasis in Cusco, Peru: A Case Series. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004361"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1850","DOI":"10.3201\/eid2707.203900","article-title":"Triclabendazole Treatment Failure for Fasciola Hepatica Infection among Preschool and School-Age Children, Cusco, Peru","volume":"27","author":"Morales","year":"2021","journal-title":"Emerg. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1016\/S0020-7519(03)00171-1","article-title":"Fasciola Hepatica Cathepsin L-like Proteases: Biology, Function, and Potential in the Development of First Generation Liver Fluke Vaccines","volume":"33","author":"Dalton","year":"2003","journal-title":"Int. J. Parasitology"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Flores-Vel\u00e1zquez, L.M., Ruiz-Campillo, M.T., Herrera-Torres, G., Mart\u00ednez-Moreno, \u00c1., Mart\u00ednez-Moreno, F.J., Zafra, R., Buffoni, L., Rufino-Moya, P.J., Molina-Hern\u00e1ndez, V., and P\u00e9rez, J. (2023). Fasciolosis: Pathogenesis, Host-Parasite Interactions, and Implication in Vaccine Development. Front. Vet. Sci., 10.","DOI":"10.3389\/fvets.2023.1270064"},{"key":"ref_16","first-page":"2074","article-title":"Fasciolosis: Recent Update in Vaccines Development and Their Efficacy","volume":"43","author":"Elsaid","year":"2023","journal-title":"Pak. Vet. J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2763","DOI":"10.1098\/rstb.2009.0089","article-title":"Zoonotic Helminth Infections with Particular Emphasis on Fasciolosis and Other Trematodiases","volume":"364","author":"Robinson","year":"2009","journal-title":"Philos. Trans. R. Soc. B Bio. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.exppara.2018.09.005","article-title":"Potential of Recombinant 2-Cys Peroxiredoxin Protein as a Vaccine for Fasciola gigantica Infection","volume":"194","author":"Sangpairoj","year":"2018","journal-title":"Exp. Parasitol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5066","DOI":"10.1128\/iai.64.12.5066-5074.1996","article-title":"Induction of Protective Immunity in Cattle against Infection with Fasciola hepatica by Vaccination with Cathepsin L Proteinases and with Hemoglobin","volume":"64","author":"Dalton","year":"1996","journal-title":"Infect. Immun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.vetpar.2016.12.009","article-title":"Characterization and Vaccine Potential of Fasciola gigantica Saposin-like Protein 1 (SAP-1)","volume":"233","author":"Kueakhai","year":"2017","journal-title":"Vet. Parasitol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5518","DOI":"10.1016\/j.vaccine.2013.09.027","article-title":"Vaccine Potential of Recombinant Saposin-like Protein 2 against Fasciolosis gigantica in Mice","volume":"31","author":"Kueakhai","year":"2013","journal-title":"Vaccine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.actatropica.2016.06.020","article-title":"Cytosolic Superoxide Dismutase Can Provide Protection against Fasciola gigantica","volume":"162","author":"Jaikua","year":"2016","journal-title":"Acta Trop."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/j.exppara.2008.01.014","article-title":"Fasciola gigantica and Schistosoma mansoni: Vaccine Potential of Recombinant Glutathione S-Transferase (RFgGST26) against Infections in Mice","volume":"119","author":"Preyavichyapugdee","year":"2008","journal-title":"Exp. Parasitol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1007\/s00436-011-2507-0","article-title":"Vaccination of Buffaloes with Fasciola gigantica Recombinant Glutathione S-Transferase and Fatty Acid Binding Protein","volume":"110","author":"Kumar","year":"2012","journal-title":"Parasitol. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1645\/GE-2891.1","article-title":"Adaptive Immune Stimulation Is Required to Obtain High Protection with Fatty Acid Binding Protein Vaccine Candidate against Fasciola hepatica in Balb\/C Mice","volume":"98","author":"Esteban","year":"2012","journal-title":"J. Parasitol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3653","DOI":"10.1007\/s00436-013-3553-6","article-title":"Immunization with Recombinant Leucine Aminopeptidase Showed Protection against Fasciola gigantica in Mice","volume":"112","author":"Changklungmoa","year":"2013","journal-title":"Parasitol. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.actatropica.2011.02.008","article-title":"Lack of Protective Efficacy in Buffaloes Vaccinated with Fasciola gigantica Leucine Aminopeptidase and Peroxiredoxin Recombinant Proteins","volume":"118","author":"Raina","year":"2011","journal-title":"Acta Trop."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1954","DOI":"10.1128\/IAI.67.4.1954-1961.1999","article-title":"Vaccination with Cathepsin L Proteinases and with Leucine Aminopeptidase Induces High Levels of Protection against Fascioliasis in Sheep","volume":"67","author":"Piacenza","year":"1999","journal-title":"Infect. Immun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5551","DOI":"10.1016\/j.vaccine.2010.06.039","article-title":"Protection of Cattle against a Natural Infection of Fasciola Hepatica by Vaccination with Recombinant Cathepsin L1 (RFhCL1)","volume":"28","author":"Golden","year":"2010","journal-title":"Vaccine"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1007\/s00436-013-3650-6","article-title":"Cathepsin L1 Mimotopes with Adjuvant Quil A Induces a Th1\/Th2 Immune Response and Confers Significant Protection against Fasciola hepatica Infection in Goats","volume":"113","year":"2014","journal-title":"Parasitol. Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.vaccine.2015.02.010","article-title":"Protection against Fasciola gigantica Infection in Mice by Vaccination with Recombinant Juvenile-Specific Cathepsin L.","volume":"33","author":"Sansri","year":"2015","journal-title":"Vaccine"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"102353","DOI":"10.1016\/j.parint.2021.102353","article-title":"The Combined Recombinant Cathepsin L1H and Cathepsin B3 Vaccine against Fasciola gigantica Infection","volume":"83","author":"Kueakhai","year":"2021","journal-title":"Parasitol. Int."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.vetpar.2016.07.009","article-title":"Vaccine Potential of Recombinant CathepsinL1G against Fasciola gigantica in Mice","volume":"226","author":"Changklungmoa","year":"2016","journal-title":"Vet. Parasitol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.actatropica.2015.06.020","article-title":"Vaccine Potential of Recombinant Pro- and Mature CathepsinL1 against Fasciolosis gigantica in Mice","volume":"150","author":"Kueakhai","year":"2015","journal-title":"Acta Trop."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.exppara.2013.06.010","article-title":"Vaccine Potential of Recombinant Cathepsin B against Fasciola gigantica","volume":"135","author":"Chantree","year":"2013","journal-title":"Exp. Parasitol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/S0065-308X(08)60243-2","article-title":"Proteinases and Associated Genes of Parasitic Helminths","volume":"43","author":"Tort","year":"1999","journal-title":"Adv. Parasitol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.molbiopara.2004.02.010","article-title":"Molecular Cloning and Analysis of Stage and Tissue-Specific Expression of Cathepsin B Encoding Genes from Fasciola gigantica","volume":"136","author":"Meemon","year":"2004","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.actatropica.2009.07.016","article-title":"Identification, Expression and Immunolocalization of Cathepsin B3, a Stage-Specific Antigen Expressed by Juvenile Fasciola gigantica","volume":"112","author":"Sethadavit","year":"2009","journal-title":"Acta Trop."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.exppara.2012.07.011","article-title":"Characterization and Expression of Cathepsin B2 in Fasciola gigantica","volume":"132","author":"Chantree","year":"2012","journal-title":"Exp. Parasitol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Montaner, S., Galiano, A., Trelis, M., Martin-Jaular, L., del Portillo, H.A., Bernal, D., and Marcilla, A. (2014). The Role of Extracellular Vesicles in Modulating the Host Immune Response during Parasitic Infections. Front. Immunol., 5.","DOI":"10.3389\/fimmu.2014.00433"},{"key":"ref_41","first-page":"234","article-title":"Antibody-Dependent Cell-Mediated Cytotoxicity against Parasites","volume":"31","author":"Capron","year":"1982","journal-title":"Prog. Allergy"},{"key":"ref_42","first-page":"30","article-title":"New Generation Vaccines for COVID-19 Based on Peptide, Viral Vector, Artificial Antigen Presenting Cell, DNA or MRNA","volume":"14","author":"Rezaei","year":"2022","journal-title":"Avicenna J. Med. Biotechnol."},{"key":"ref_43","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":"2020","journal-title":"Chem. Rev."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"842","DOI":"10.1039\/C5SC03892H","article-title":"Peptide-Based Synthetic Vaccines","volume":"7","author":"Skwarczynski","year":"2016","journal-title":"Chem. Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1111\/j.1529-8019.2009.01223.x","article-title":"Vaccine Immunology","volume":"22","author":"Bartlett","year":"2009","journal-title":"Dermatol. Ther."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Rastogi, I., Jeon, D., Moseman, J.E., Muralidhar, A., Potluri, H.K., and McNeel, D.G. (2022). Role of B Cells as Antigen Presenting Cells. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.954936"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1007\/s10989-023-10535-0","article-title":"Advances in Computational and Bioinformatics Tools and Databases for Designing and Developing a Multi-Epitope-Based Peptide Vaccine","volume":"29","author":"Shawan","year":"2023","journal-title":"Int. J. Pept. Res. Ther."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1016\/j.molimm.2019.04.021","article-title":"The Schistosoma mansoni Cyclophilin A Epitope 107-121 Induces a Protective Immune Response against Schistosomiasis","volume":"111","author":"Mendes","year":"2019","journal-title":"Mol. Immunol."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Gazzinelli-Guimar\u00e3es, A.C., Nogueira, D.S., Amorim, C.C.O., Oliveira, F.M.S., Coqueiro-Dos-Santos, A., Carvalho, S.A.P., Kraemer, L., Barbosa, F.S., Fraga, V.G., and Santos, F.V. (2021). ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.788185"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Zawawi, A., Forman, R., Smith, H., Mair, I., Jibril, M., Albaqshi, M.H., Brass, A., Derrick, J.P., and Else, K.J. (2020). In Silico Design of a T-Cell Epitope Vaccine Candidate for Parasitic Helminth Infection. PLoS Pathog., 16.","DOI":"10.1371\/journal.ppat.1008243"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"2074803","DOI":"10.1155\/2020\/2074803","article-title":"A Multiple Antigen Peptide Vaccine Containing CD4+ T Cell Epitopes Enhances Humoral Immunity against Trichinella spiralis Infection in Mice","volume":"2020","author":"Gu","year":"2020","journal-title":"J. Immunol. Res."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Chansap, S., Cheukamud, W., Suthisintong, T., Kueakhai, P., and Changklungmoa, N. (2025). Development of Multi-Epitope Cathepsin L Driven Short Peptide Vaccine against Fasciola gigantica. Front. Vet. Sci., 12.","DOI":"10.3389\/fvets.2025.1547937"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Rojas-Caraballo, J., L\u00f3pez-Ab\u00e1n, J., P\u00e9rez Del Villar, L., Vizca\u00edno, C., Vicente, B., Fern\u00e1ndez-Soto, P., Del Olmo, E., Patarroyo, M.A., and Muro, A. (2014). In Vitro and in Vivo Studies for Assessing the Immune Response and Protection-Inducing Ability Conferred by Fasciola Hepatica-Derived Synthetic Peptides Containing B- and T-Cell Epitopes. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0105323"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/s00430-019-00640-7","article-title":"A New Multi-Epitope Peptide Vaccine Induces Immune Responses and Protection against Leishmania infantum in BALB\/c Mice","volume":"209","author":"Vakili","year":"2020","journal-title":"Med. Microbiol. Immunol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Guo, L., Yang, H., Tang, F., Yin, R., Liu, H., Gong, X., Wei, J., Zhang, Y., Xu, G., and Liu, K. (2017). Oral Immunization with a Multivalent Epitope-Based Vaccine, Based on NAP, Urease, HSP60, and HpaA, Provides Therapeutic Effect on H. pylori Infection in Mongolian Gerbils. Front. Cell. Infect. Microbiol., 7.","DOI":"10.3389\/fcimb.2017.00349"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1080\/22221751.2021.1921621","article-title":"A Novel Linker-Immunodominant Site (LIS) Vaccine Targeting the SARS-CoV-2 Spike Protein Protects against Severe COVID-19 in Syrian hamsters","volume":"10","author":"Zhang","year":"2021","journal-title":"Emerg. Microbes Infect."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"2621","DOI":"10.1016\/j.immuni.2023.10.005","article-title":"Leveraging Vaccination-Induced Protective Antibodies to Define Conserved Epitopes on Influenza N2 Neuraminidase","volume":"56","author":"Lei","year":"2023","journal-title":"Immunity"},{"key":"ref_58","doi-asserted-by":"crossref","unstructured":"Rapin, N., Lund, O., Bernaschi, M., and Castiglione, F. (2010). Computational Immunology Meets Bioinformatics: The Use of Prediction Tools for Molecular Binding in the Simulation of the Immune System. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0009862"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"4064","DOI":"10.1016\/j.vaccine.2017.06.003","article-title":"The Complexity and Cost of Vaccine Manufacturing\u2014An Overview","volume":"35","author":"Plotkin","year":"2017","journal-title":"Vaccine"},{"key":"ref_60","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":"Raoufi","year":"2020","journal-title":"Int. J. Pept. Res. Ther."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1021\/acsabm.1c01238","article-title":"Peptides for Vaccine Development","volume":"5","author":"Hamley","year":"2022","journal-title":"ACS Appl. Bio. Mater."},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Ge, H., Sun, L., and Yu, J. (2017). Fast Batch Searching for Protein Homology Based on Compression and Clustering. BMC Bioinform., 18.","DOI":"10.1186\/s12859-017-1938-8"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1016\/j.vaccine.2018.12.005","article-title":"Would Immunization Be the Same without Cross-Reactivity?","volume":"37","author":"Vojtek","year":"2019","journal-title":"Vaccine"},{"key":"ref_64","doi-asserted-by":"crossref","unstructured":"Trier, N.H., and Houen, G. (2023). Antibody Cross-Reactivity in Auto-Immune Diseases. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241713609"},{"key":"ref_65","doi-asserted-by":"crossref","unstructured":"Schaap-Johansen, A.L., Vujovi\u0107, M., Borch, A., Hadrup, S.R., and Marcatili, P. (2021). T Cell Epitope Prediction and Its Application to Immunotherapy. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.712488"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1002\/pro.2206","article-title":"Linkers in the Structural Biology of Protein-Protein Interactions","volume":"22","author":"Kumar","year":"2013","journal-title":"Protein Sci."},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Khan, M., Khan, S., Ali, A., Akbar, H., Sayaf, A.M., Khan, A., and Wei, D.Q. (2019). Immunoinformatics Approaches to Explore Helicobacter Pylori Proteome (Virulence Factors) to Design B and T Cell Multi-Epitope Subunit Vaccine. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-49354-z"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"25151355221100218","DOI":"10.1177\/25151355221100218","article-title":"Predicting Epitopes for Vaccine Development Using Bioinformatics Tools","volume":"10","author":"Yurina","year":"2022","journal-title":"Ther. Adv. Vaccines Immunother."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1016\/j.addr.2012.09.039","article-title":"Fusion Protein Linkers: Property, Design and Functionality","volume":"65","author":"Chen","year":"2013","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"2963","DOI":"10.1080\/07391102.2020.1850357","article-title":"Design of a Multi-Epitope-Based Vaccine Targeting M-Protein of SARS-CoV2: An Immunoinformatics Approach","volume":"40","author":"Ayyagari","year":"2022","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"586","DOI":"10.1038\/cmi.2017.151","article-title":"Vaccine-Induced Autoimmunity: The Role of Molecular Mimicry and Immune Crossreaction","volume":"15","author":"Segal","year":"2018","journal-title":"Cell. Mol. Immunol."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"339","DOI":"10.2174\/0929866526666190228144219","article-title":"Applicability of Instability Index for In Vitro Protein Stability Prediction","volume":"26","author":"Gamage","year":"2019","journal-title":"Protein Pept. Lett."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"S3","DOI":"10.1016\/j.jaci.2009.12.980","article-title":"Overview of the Immune Response","volume":"125","author":"Chaplin","year":"2010","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_74","doi-asserted-by":"crossref","unstructured":"Wiltgen, M. (2018). Algorithms for Structure Comparison and Analysis: Homology Modelling of Proteins. Encyclopedia of Bioinformatics and Computational Biology: ABC of Bioinformatics, Elsevier.","DOI":"10.1016\/B978-0-12-809633-8.20484-6"},{"key":"ref_75","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_76","doi-asserted-by":"crossref","unstructured":"Park, S.W., Lee, B.H., Song, S.H., and Kim, M.K. (2023). Revisiting the Ramachandran Plot Based on Statistical Analysis of Static and Dynamic Characteristics of Protein Structures. J. Struct. Biol., 215.","DOI":"10.1016\/j.jsb.2023.107939"},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1016\/j.molimm.2019.09.014","article-title":"B-Cell Epitopes: Discontinuity and Conformational Analysis","volume":"114","author":"Ferdous","year":"2019","journal-title":"Mol. Immunol."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"2735","DOI":"10.1002\/jmv.26826","article-title":"Role of Toll-like Receptors in the Pathogenesis of COVID-19","volume":"93","author":"Khanmohammadi","year":"2021","journal-title":"J. Med. Virol."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.ijbiomac.2019.07.024","article-title":"Development of Multi-Epitope Driven Subunit Vaccine against Fasciola gigantica Using Immunoinformatics Approach","volume":"138","author":"Kalita","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"619410","DOI":"10.1155\/2014\/619410","article-title":"Targeting TLR2 for Vaccine Development","volume":"2014","author":"Basto","year":"2014","journal-title":"J. Immunol. Res."},{"key":"ref_81","doi-asserted-by":"crossref","unstructured":"Oliveira-Nascimento, L., Massari, P., and Wetzler, L.M. (2012). The Role of TLR2 Ininfection and Immunity. Front. Immunol., 3.","DOI":"10.3389\/fimmu.2012.00079"},{"key":"ref_82","doi-asserted-by":"crossref","unstructured":"Das, K.C., Konhar, R., and Biswal, D.K. (2023). Fasciola gigantica Vaccine Construct: An in Silico Approach towards Identification and Design of a Multi-Epitope Subunit Vaccine Using Calcium Binding EF-Hand Proteins. BMC Immunol., 24.","DOI":"10.1186\/s12865-022-00535-y"},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"104352","DOI":"10.1016\/j.meegid.2020.104352","article-title":"Designing a Vaccine for Fascioliasis Using Immunogenic 24 KDa Mu-Class Glutathione s-Transferase","volume":"83","author":"Kalita","year":"2020","journal-title":"Infect. Genet. Evol."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1080\/14760584.2022.1996233","article-title":"Construction of a Multiepitope Vaccine Candidate against Fasciola Hepatica: An in Silico Design Using Various Immunogenic Excretory\/Secretory Antigens","volume":"21","author":"Aykur","year":"2022","journal-title":"Expert Rev. Vaccines"},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"W271","DOI":"10.1093\/nar\/gku339","article-title":"IMODS: Internal Coordinates Normal Mode Analysis Server","volume":"42","author":"Aliaga","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_86","doi-asserted-by":"crossref","unstructured":"Sanches, R.C.O., Tiwari, S., Ferreira, L.C.G., Oliveira, F.M., Lopes, M.D., Passos, M.J.F., Maia, E.H.B., Taranto, A.G., Kato, R., and Azevedo, V.A.C. (2021). Immunoinformatics Design of Multi-Epitope Peptide-Based Vaccine Against Schistosoma mansoni Using Transmembrane Proteins as a Target. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.621706"},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"113475","DOI":"10.1016\/j.jim.2023.113475","article-title":"A Multi-Epitope Based Vaccine against the Surface Proteins Expressed in Cyst and Trophozoite Stages of Parasite Entamoeba histolytica","volume":"517","author":"Chatterjee","year":"2023","journal-title":"J. Immunol. Methods"},{"key":"ref_88","doi-asserted-by":"crossref","unstructured":"Qureshi, R., and Qureshi, I.A. (2023). Development of a Multi-Epitope Vaccine Candidate for Leishmanial Parasites Applying Immunoinformatics and in Vitro Approaches. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1269774"},{"key":"ref_89","doi-asserted-by":"crossref","unstructured":"Saha, S., Vashishtha, S., Kundu, B., and Ghosh, M. (2022). In-Silico Design of an Immunoinformatics Based Multi-Epitope Vaccine against Leishmania donovani. BMC Bioinform., 23.","DOI":"10.1186\/s12859-022-04816-6"},{"key":"ref_90","doi-asserted-by":"crossref","unstructured":"Sarfraz, A., Wara, T.U., Chen, K., Ansari, S.H., Zaman, A., Nishan, U., Iqbal, A., Ullah, R., and Ali, E.A. (2023). Structural Informatics Approach for Designing an Epitope-Based Vaccine against the Brain-Eating Naegleria fowleri. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1284621"},{"key":"ref_91","doi-asserted-by":"crossref","unstructured":"Aziz, S., Waqas, M., Halim, S.A., Ali, A., Iqbal, A., Iqbal, M., Khan, A., and Al-Harrasi, A. (2022). Exploring Whole Proteome to Contrive Multi-Epitope-Based Vaccine for NeoCoV: An Immunoinformtics and in-Silico Approach. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.956776"},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.vetpar.2013.04.008","article-title":"Immunomodulatory Molecules of Fasciola hepatica: Candidates for Both Vaccine and Immunotherapeutic Development","volume":"195","author":"Dalton","year":"2013","journal-title":"Vet. Parasitol."},{"key":"ref_93","doi-asserted-by":"crossref","unstructured":"Meira, C.d.S., and Gedamu, L. (2019). Protective or Detrimental? Understanding the Role of Host Immunity in Leishmaniasis. Microorganisms, 7.","DOI":"10.3390\/microorganisms7120695"},{"key":"ref_94","doi-asserted-by":"crossref","unstructured":"Liang, S., Zhang, S., Bao, Y., Zhang, Y., Liu, X., Yao, H., and Liu, G. (2024). Combined Immunoinformatics to Design and Evaluate a Multi-Epitope Vaccine Candidate against Streptococcus suis Infection. Vaccines, 12.","DOI":"10.3390\/vaccines12020137"},{"key":"ref_95","doi-asserted-by":"crossref","unstructured":"Umar, A., Haque, A., Alghamdi, Y.S., Mashraqi, M.M., Rehman, A., Shahid, F., Khurshid, M., and Ashfaq, U.A. (2021). Development of a Candidate Multi-Epitope Subunit Vaccine against Klebsiella aerogenes: Subtractive Proteomics and Immuno-Informatics Approach. Vaccines, 9.","DOI":"10.3390\/vaccines9111373"},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1111\/j.0818-9641.2004.01272.x","article-title":"Vaccine Adjuvants: Current State and Future Trends","volume":"82","author":"Petrovsky","year":"2004","journal-title":"Immunol. Cell Biol."},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/S0020-7519(03)00053-5","article-title":"Recent Advances in Veterinary Vaccine Adjuvants","volume":"33","author":"Singh","year":"2003","journal-title":"Int. J. Parasitol."},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"1597","DOI":"10.1038\/nm.3409","article-title":"Key Roles of Adjuvants in Modern Vaccines","volume":"19","author":"Reed","year":"2013","journal-title":"Nat. Med."},{"key":"ref_99","doi-asserted-by":"crossref","unstructured":"Bian, L., Zheng, Y., Guo, X., Li, D., Zhou, J., Jing, L., Chen, Y., Lu, J., Zhang, K., and Jiang, C. (2022). Intramuscular Inoculation of AS02-Adjuvanted Respiratory Syncytial Virus (RSV) F Subunit Vaccine Shows Better Efficiency and Safety Than Subcutaneous Inoculation in BALB\/c Mice. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.938598"},{"key":"ref_100","doi-asserted-by":"crossref","first-page":"7659684","DOI":"10.1155\/2016\/7659684","article-title":"Chitosan and Sodium Alginate Combinations Are Alternative, Efficient, and Safe Natural Adjuvant Systems for Hepatitis B Vaccine in Mouse Model","volume":"2016","author":"Abdelallah","year":"2016","journal-title":"Evid.-Based Complement. Altern. Med."},{"key":"ref_101","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.exppara.2015.11.002","article-title":"Adjuvant-Enhanced Antibody and Cellular Responses to Inclusion Bodies Expressing FhSAP2 Correlates with Protection of Mice to Fasciola hepatica","volume":"160","author":"Rivera","year":"2016","journal-title":"Exp. Parasitol."},{"key":"ref_102","doi-asserted-by":"crossref","unstructured":"Mielke, D., Bandawe, G., Pollara, J., Abrahams, M.R., Nyanhete, T., Moore, P.L., Thebus, R., Yates, N.L., Kappes, J.C., and Ochsenbauer, C. (2019). Antibody-Dependent Cellular Cytotoxicity (ADCC)-Mediating Antibodies Constrain Neutralizing Antibody Escape Pathway. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.02875"},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"a007161","DOI":"10.1101\/cshperspect.a007161","article-title":"HIV-1 Antiretroviral Drug Therapy","volume":"2","author":"Arts","year":"2012","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_104","doi-asserted-by":"crossref","unstructured":"Gro\u00df, A., Hashimoto, C., Sticht, H., and Eichler, J. (2016). Synthetic Peptides as Protein Mimics. Front. Bioeng. Biotechnol., 3.","DOI":"10.3389\/fbioe.2015.00211"},{"key":"ref_105","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_106","doi-asserted-by":"crossref","unstructured":"Doytchinova, I.A., and Flower, D.R. (2007). VaxiJen: A Server for Prediction of Protective Antigens, Tumour Antigens and Subunit Vaccines. BMC Bioinform., 8.","DOI":"10.1186\/1471-2105-8-4"},{"key":"ref_107","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1093\/bioinformatics\/btt619","article-title":"AllergenFP: Allergenicity Prediction by Descriptor Fingerprints","volume":"30","author":"Dimitrov","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"W449","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":"Reynisson","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_109","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1007\/978-1-4939-2285-7_7","article-title":"Peptide Toxicity Prediction","volume":"1268","author":"Gupta","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_110","doi-asserted-by":"crossref","first-page":"1265","DOI":"10.1038\/s41467-022-28865-w","article-title":"Improved Prediction of Protein-Protein Interactions Using AlphaFold2","volume":"13","author":"Bryant","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_111","doi-asserted-by":"crossref","first-page":"W384","DOI":"10.1093\/nar\/gkt458","article-title":"GalaxyRefine: Protein Structure Refinement Driven by Side-Chain Repacking","volume":"41","author":"Heo","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_112","doi-asserted-by":"crossref","unstructured":"Ponomarenko, J., Bui, H.H., Li, W., Fusseder, N., Bourne, P.E., Sette, A., and Peters, B. (2008). ElliPro: A New Structure-Based Tool for the Prediction of Antibody Epitopes. BMC Bioinform., 9.","DOI":"10.1186\/1471-2105-9-514"},{"key":"ref_113","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1002\/prot.25219","article-title":"New Additions to the ClusPro Server Motivated by CAPRI","volume":"85","author":"Vajda","year":"2017","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_114","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein Measurement with the Folin Phenol Reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/14\/6971\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:12:57Z","timestamp":1760033577000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/14\/6971"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,20]]},"references-count":114,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["ijms26146971"],"URL":"https:\/\/doi.org\/10.3390\/ijms26146971","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,20]]}}}