{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:15:39Z","timestamp":1787030139173,"version":"build-2736575974"},"reference-count":44,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,1,17]],"date-time":"2025-01-17T00:00:00Z","timestamp":1737072000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&amp;D Program of China","doi-asserted-by":"publisher","award":["2022YFC2303402"],"award-info":[{"award-number":["2022YFC2303402"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Polypeptide vaccines have the potential to improve immune responses by targeting conserved and weakly immunogenic regions in antigens. This study aimed to identify and evaluate the efficacy of a novel influenza universal vaccine candidate consisting of multiple polypeptides derived from highly conserved regions of influenza virus proteins hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). Methods: Immunoinformatics tools were used to screen conserved epitopes from different influenza virus subtypes (H1N1, H3N2, H5N1, H7N9, H9N2, and IBV). A polypeptide vaccine, P125-H, was constructed by linking multiple epitopes using Ii-Key technology. The immunogenicity of P125-H was assessed in mice using MF59-adjuvanted P125-H via intraperitoneal injection. Hemagglutination inhibition (HI) and neutralizing antibody responses were measured, along with IFN-\u03b3 levels in spleen lymphocytes. Protective efficacy was evaluated using viral challenge with lethal doses of H1N1 and H7N9. Results: Mice immunized with P125-H generated high levels of HI and neutralizing antibodies against multiple influenza strains. IFN-\u03b3 production was significantly elevated in spleen lymphocytes upon stimulation with the vaccine. P125-H protected mice from influenza infection, reducing weight loss and the viral load in the lungs, mitigating lung pathology, and decreasing mortality. Conclusions: The P125-H vaccine induced broad cross-protection against multiple influenza strains and elicited robust immune responses. It demonstrates strong potential as a candidate for a universal influenza vaccine.<\/jats:p>","DOI":"10.3390\/vaccines13010081","type":"journal-article","created":{"date-parts":[[2025,1,17]],"date-time":"2025-01-17T03:33:02Z","timestamp":1737084782000},"page":"81","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["The Development of a Novel Broad-Spectrum Influenza Polypeptide Vaccine Based on Multi-Epitope Tandem Sequences"],"prefix":"10.3390","volume":"13","author":[{"given":"Song","family":"Zhao","sequence":"first","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junhao","family":"Luo","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenhui","family":"Guo","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Li","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyu","family":"Pu","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Libo","family":"Dong","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenfei","family":"Zhu","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1139-3607","authenticated-orcid":false,"given":"Rongbao","family":"Gao","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"},{"name":"NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e203","DOI":"10.1016\/S2666-5247(23)00013-7","article-title":"The influenza imperative: We must prepare now for seasonal and pandemic influenza","volume":"4","author":"Dzau","year":"2023","journal-title":"Lancet. Microbe"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1001\/jamacardio.2016.0433","article-title":"Seasonal Influenza Infections and Cardiovascular Disease Mortality","volume":"1","author":"Nguyen","year":"2016","journal-title":"JAMA Cardiol."},{"key":"ref_3","unstructured":"Word Health Organization (2023, October 03). Influenza (Seasonal). Available online: https:\/\/www.who.int\/en\/news-room\/fact-sheets\/detail\/influenza-(seasonal)."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"D49","DOI":"10.1016\/j.vaccine.2008.07.039","article-title":"The biology of influenza viruses","volume":"26","author":"Bouvier","year":"2008","journal-title":"Vaccine"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"S50","DOI":"10.1038\/d41586-019-02751-w","article-title":"Towards a universal flu vaccine","volume":"573","author":"Eisenstein","year":"2019","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e902","DOI":"10.1016\/S2666-5247(22)00293-2","article-title":"Universal flu vaccine protects against influenza A and B","volume":"3","author":"Feranmi","year":"2022","journal-title":"Lancet. Microbe"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Zhu, W., Wang, C., and Wang, B.Z. (2017). From Variation of Influenza Viral Proteins to Vaccine Development. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18071554"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1141","DOI":"10.1080\/14760584.2017.1379396","article-title":"Vaccine approaches conferring cross-protection against influenza viruses","volume":"16","author":"Vemula","year":"2017","journal-title":"Expert Rev. Vaccines"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1016\/j.ijbiomac.2022.12.066","article-title":"Designing multi-epitope mRNA construct as a universal influenza vaccine candidate for future epidemic\/pandemic preparedness","volume":"226","author":"Rcheulishvili","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"105095","DOI":"10.1016\/j.micpath.2021.105095","article-title":"Bursal peptide BP-IV as a novel immunoadjuvant enhances the protective efficacy of an epitope peptide vaccine containing T and B cell epitopes of the H9N2 avian influenza virus","volume":"158","author":"Liu","year":"2021","journal-title":"Microb. Pathog."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sanchez, P.L., Staats, H.F., Abraham, S.N., and Ross, T.M. (2024). Mastoparan-7 adjuvanted COBRA H1 and H3 hemagglutinin influenza vaccines. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-64351-7"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"515","DOI":"10.3390\/vaccines2030515","article-title":"Peptide Vaccine: Progress and Challenges","volume":"2","author":"Li","year":"2014","journal-title":"Vaccines"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Bullard, B.L., and Weaver, E.A. (2021). Strategies Targeting Hemagglutinin as a Universal Influenza Vaccine. Vaccines, 9.","DOI":"10.3390\/vaccines9030257"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1126\/science.1204839","article-title":"A highly conserved neutralizing epitope on group 2 influenza A viruses","volume":"333","author":"Ekiert","year":"2011","journal-title":"Science"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5593","DOI":"10.1038\/s41467-024-49884-9","article-title":"A(H2N2) and A(H3N2) influenza pandemics elicited durable cross-reactive and protective antibodies against avian N2 neuraminidases","volume":"15","author":"Liang","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1126\/science.aay0678","article-title":"Broadly protective human antibodies that target the active site of influenza virus neuraminidase","volume":"366","author":"Stadlbauer","year":"2019","journal-title":"Science"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"S68","DOI":"10.1093\/infdis\/jiz003","article-title":"The Role of Matrix Protein 2 Ectodomain in the Development of Universal Influenza Vaccines","volume":"219","author":"Saelens","year":"2019","journal-title":"J. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"23545","DOI":"10.1021\/acsnano.3c06526","article-title":"Single-Component Multilayered Self-Assembling Protein Nanoparticles Displaying Extracellular Domains of Matrix Protein 2 as a Pan-influenza A Vaccine","volume":"17","author":"Zhang","year":"2023","journal-title":"ACS Nano"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1080\/22221751.2018.1558962","article-title":"Protection against homo and hetero-subtypic influenza A virus by optimized M2e DNA vaccine","volume":"8","author":"Yao","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1311","DOI":"10.1517\/14712598.6.12.1311","article-title":"Ii-Key\/MHC class II epitope hybrids: A strategy that enhances MHC class II epitope loading to create more potent peptide vaccines","volume":"6","author":"Kallinteris","year":"2006","journal-title":"Expert Opin. Biol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2805","DOI":"10.1016\/j.vaccine.2012.02.031","article-title":"CD4+ T-cell activation for immunotherapy of malignancies using Ii-Key\/MHC class II epitope hybrid vaccines","volume":"30","author":"Xu","year":"2012","journal-title":"Vaccine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1080\/14760584.2019.1622419","article-title":"Challenges in estimating influenza vaccine effectiveness","volume":"18","author":"Ainslie","year":"2019","journal-title":"Expert Rev. Vaccines"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"168","DOI":"10.15585\/mmwr.mm7308a3","article-title":"Interim Estimates of 2023-24 Seasonal Influenza Vaccine Effectiveness\u2014United States","volume":"73","author":"Frutos","year":"2024","journal-title":"MMWR. Morb. Mortal. Wkly. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"De Groot, A.S., Moise, L., Terry, F., Gutierrez, A.H., Hindocha, P., Richard, G., Hoft, D.F., Ross, T.M., Noe, A.R., and Takahashi, Y. (2020). Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.00442"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Allen, J.D., and Ross, T.M. (2024). mRNA vaccines encoding computationally optimized hemagglutinin elicit protective antibodies against future antigenically drifted H1N1 and H3N2 influenza viruses isolated between 2018\u20132020. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1334670"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Allen, J.D., and Ross, T.M. (2021). Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses. Sci. Rep., 11.","DOI":"10.1038\/s41598-020-79590-7"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"100768","DOI":"10.1016\/j.imu.2021.100768","article-title":"Role of artificial intelligence in peptide vaccine design against RNA viruses","volume":"26","author":"Mohanty","year":"2021","journal-title":"Inform. Med. Unlocked"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2716","DOI":"10.1016\/j.vaccine.2023.03.023","article-title":"Safety and immunogenicity of Multimeric-001 (M-001) followed by seasonal quadrivalent inactivated influenza vaccine in young adults\u2014A randomized clinical trial","volume":"41","author":"Atmar","year":"2023","journal-title":"Vaccine"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1038\/s41541-020-0174-9","article-title":"Efficacy of FLU-v, a broad-spectrum influenza vaccine, in a randomized phase IIb human influenza challenge study","volume":"5","author":"Pleguezuelos","year":"2020","journal-title":"NPJ Vaccines"},{"key":"ref_30","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_31","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.it.2023.11.001","article-title":"Targeting neuraminidase: The next frontier for broadly protective influenza vaccines","volume":"45","author":"Wu","year":"2024","journal-title":"Trends Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/nsmb.1566","article-title":"Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses","volume":"16","author":"Sui","year":"2009","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4128","DOI":"10.1016\/S0264-410X(03)00493-6","article-title":"Ii-Key\/MHC class II epitope hybrid peptide vaccines for HIV","volume":"21","author":"Kallinteris","year":"2003","journal-title":"Vaccine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.smim.2018.05.001","article-title":"Correlates of adjuvanticity: A review on adjuvants in licensed vaccines","volume":"39","author":"Rappuoli","year":"2018","journal-title":"Semin. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Akhtar, N., Kaushik, V., Grewal, R.K., Wani, A.K., Suwattanasophon, C., Choowongkomon, K., Oliva, R., Shaikh, A.R., Cavallo, L., and Chawla, M. (2022). Immunoinformatics-Aided Design of a Peptide Based Multiepitope Vaccine Targeting Glycoproteins and Membrane Proteins against Monkeypox Virus. Viruses, 14.","DOI":"10.3390\/v14112374"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Sharif, N., Alzahrani, K.J., Ahmed, S.N., and Dey, S.K. (2021). Efficacy, Immunogenicity and Safety of COVID-19 Vaccines: A Systematic Review and Meta-Analysis. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.714170"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.smim.2018.08.002","article-title":"Correlating efficacy and immunogenicity in malaria vaccine trials","volume":"39","author":"McCall","year":"2018","journal-title":"Semin. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"899","DOI":"10.1126\/science.abm0271","article-title":"A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes","volume":"378","author":"Arevalo","year":"2022","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1141","DOI":"10.1016\/j.immuni.2024.03.022","article-title":"Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses","volume":"57","author":"Ray","year":"2024","journal-title":"Immunity"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1615\/CritRevImmunol.2013010019","article-title":"T-cell immunity to influenza A viruses","volume":"34","author":"Grant","year":"2014","journal-title":"Crit. Rev. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3183","DOI":"10.1182\/blood.V95.10.3183","article-title":"Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4","volume":"95","author":"Wang","year":"2000","journal-title":"Blood"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1038\/mt.2012.246","article-title":"Virus-like particles containing multiple M2 extracellular domains confer improved cross-protection against various subtypes of influenza virus","volume":"21","author":"Kim","year":"2013","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Bliss, C.M., Nachbagauer, R., Mariottini, C., Cuevas, F., Feser, J., Naficy, A., Bernstein, D.I., Guptill, J., Walter, E.B., and Berlanda-Scorza, F. (2024). A chimeric haemagglutinin-based universal influenza virus vaccine boosts human cellular immune responses directed towards the conserved haemagglutinin stalk domain and the viral nucleoprotein. EBioMedicine, 104.","DOI":"10.1016\/j.ebiom.2024.105153"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Carascal, M.B., Pavon, R.D.N., and Rivera, W.L. (2022). Recent Progress in Recombinant Influenza Vaccine Development Toward Heterosubtypic Immune Response. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.878943"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/1\/81\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T10:30:18Z","timestamp":1759919418000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/1\/81"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,17]]},"references-count":44,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["vaccines13010081"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13010081","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,17]]}}}