{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T22:08:07Z","timestamp":1783462087100,"version":"3.55.0"},"reference-count":37,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,7,11]],"date-time":"2025-07-11T00:00:00Z","timestamp":1752192000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32472298"],"award-info":[{"award-number":["32472298"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["ZR2024MC022"],"award-info":[{"award-number":["ZR2024MC022"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["M2024-04"],"award-info":[{"award-number":["M2024-04"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007129","name":"Shandong Provincial Natural Science Foundation","doi-asserted-by":"publisher","award":["32472298"],"award-info":[{"award-number":["32472298"]}],"id":[{"id":"10.13039\/501100007129","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007129","name":"Shandong Provincial Natural Science Foundation","doi-asserted-by":"publisher","award":["ZR2024MC022"],"award-info":[{"award-number":["ZR2024MC022"]}],"id":[{"id":"10.13039\/501100007129","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007129","name":"Shandong Provincial Natural Science Foundation","doi-asserted-by":"publisher","award":["M2024-04"],"award-info":[{"award-number":["M2024-04"]}],"id":[{"id":"10.13039\/501100007129","id-type":"DOI","asserted-by":"publisher"}]},{"name":"State Key Laboratory of Microbial Technology Open Projects Fund","award":["32472298"],"award-info":[{"award-number":["32472298"]}]},{"name":"State Key Laboratory of Microbial Technology Open Projects Fund","award":["ZR2024MC022"],"award-info":[{"award-number":["ZR2024MC022"]}]},{"name":"State Key Laboratory of Microbial Technology Open Projects Fund","award":["M2024-04"],"award-info":[{"award-number":["M2024-04"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli\u2019s natural capacity to protect surface-displayed antigens, as evidenced by HA1\u2019s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines.<\/jats:p>","DOI":"10.3390\/microorganisms13071649","type":"journal-article","created":{"date-parts":[[2025,7,11]],"date-time":"2025-07-11T15:19:31Z","timestamp":1752247171000},"page":"1649","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Surface Display of Avian H5 and H9 Hemagglutinin Antigens on Non-Genetically Modified Lactobacillus Cells for Bivalent Oral AIV Vaccine Development"],"prefix":"10.3390","volume":"13","author":[{"given":"Fuyi","family":"Liu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"},{"name":"School of Life Sciences, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingbo","family":"Chang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"},{"name":"School of Life Sciences, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1848-5773","authenticated-orcid":false,"given":"Jingqi","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"},{"name":"School of Life Sciences, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuping","family":"Liao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaonan","family":"Deng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingting","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2872-2078","authenticated-orcid":false,"given":"Jian","family":"Kong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3537-4099","authenticated-orcid":false,"given":"Wentao","family":"Kong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1046\/j.1365-2672.2003.01850.x","article-title":"Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract","volume":"94","author":"Boris","year":"2003","journal-title":"J. Appl. Microbiol."},{"key":"ref_2","unstructured":"O\u2019Callaghan, J., and O\u2019Toole, P.W. (2013). Lactobacillus: Host\u2013microbe relationships. Between Pathogenicity and Commensalism, Springer."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"S17","DOI":"10.1186\/1475-2859-10-S1-S17","article-title":"Immunomodulatory mechanisms of lactobacilli","volume":"10","author":"Wells","year":"2011","journal-title":"Microb. Cell Factories"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Dempsey, E., and Corr, S.C. (2022). Lactobacillus spp. for gastrointestinal health: Current and future perspectives. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.840245"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2117","DOI":"10.1016\/S0264-410X(99)00010-9","article-title":"Instruments for oral disease-intervention strategies: Recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis","volume":"17","author":"Maassen","year":"1999","journal-title":"Vaccine"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"508","DOI":"10.1016\/S0167-7799(02)02075-9","article-title":"Lactobacilli as live vaccine delivery vectors: Progress and prospects","volume":"20","author":"Seegers","year":"2002","journal-title":"Trends Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"S4","DOI":"10.1186\/1475-2859-10-S1-S4","article-title":"Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines","volume":"10","author":"Kharrat","year":"2011","journal-title":"Microb. Cell Factories"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3339","DOI":"10.1016\/j.vaccine.2011.08.036","article-title":"Immunization with recombinant Lactobacillus casei strains producing K99, K88 fimbrial protein protects mice against enterotoxigenic Escherichia coli","volume":"30","author":"Wen","year":"2012","journal-title":"Vaccine"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2967","DOI":"10.1007\/s00253-015-6498-0","article-title":"Lactic acid bacteria\u201420 years exploring their potential as live vectors for mucosal vaccination","volume":"99","author":"Kobierecka","year":"2015","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s00253-004-1851-8","article-title":"Cell surface display system for Lactococcus lactis: A novel development for oral vaccine","volume":"68","author":"Raha","year":"2005","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1046\/j.1365-2958.2002.03180.x","article-title":"Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly","volume":"46","author":"Antikainen","year":"2002","journal-title":"Mol. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"953","DOI":"10.1016\/S0022-2836(02)01135-X","article-title":"Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356: Evidence for protein\u2013protein interaction of two subdomains","volume":"324","author":"Smit","year":"2002","journal-title":"J. Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"\u00c5vall-J\u00e4\u00e4skel\u00e4inen, S., Hyn\u00f6nen, U., Ilk, N., Pum, D., Sleytr, U.B., and Palva, A. (2008). Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287. BMC Microbiol., 8.","DOI":"10.1186\/1471-2180-8-165"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1186\/1475-2859-10-86","article-title":"Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein","volume":"10","author":"Hu","year":"2011","journal-title":"Microb. Cell Factories"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1941","DOI":"10.1007\/s00253-012-4044-x","article-title":"Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen","volume":"97","author":"Sun","year":"2013","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Uriza, P.J., Trautman, C., Palomino, M.M., Fina Martin, J., Ruzal, S.M., Roset, M.S., and Briones, G. (2020). Development of an antigen delivery platform using Lactobacillus acidophilus decorated with heterologous proteins: A sheep in wolf\u2019s clothing story. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.509380"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1007\/978-1-0716-0346-8_7","article-title":"A Brief Introduction to Avian Influenza Virus","volume":"2123","author":"Spackman","year":"2020","journal-title":"Methods Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Tong, S., Zhu, X., Li, Y., Shi, M., Zhang, J., Bourgeois, M., Yang, H., Chen, X., Recuenco, S., and Gomez, J. (2013). New world bats harbor diverse influenza A viruses. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003657"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"a038612","DOI":"10.1101\/cshperspect.a038612","article-title":"Bat-borne influenza A viruses: An awakening","volume":"11","author":"Ciminski","year":"2021","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"a038778","DOI":"10.1101\/cshperspect.a038778","article-title":"Influenza hemagglutinin structures and antibody recognition","volume":"10","author":"Wu","year":"2020","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Shirvani, E., Paldurai, A., Varghese, B.P., and Samal, S.K. (2020). Contributions of HA1 and HA2 subunits of highly pathogenic avian influenza virus in induction of neutralizing antibodies and protection in chickens. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.01085"},{"key":"ref_22","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"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1186\/s12985-023-02044-9","article-title":"Vaccination with recombinant Lactococcus lactis expressing HA1-IgY Fc fusion protein provides protective mucosal immunity against H9N2 avian influenza virus in chickens","volume":"20","author":"Zhang","year":"2023","journal-title":"Virol. J."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Li, Z., Peng, C., Chen, L., Wang, P., and Wang, F. (2024). Construction and Immunogenicity Evaluation of Recombinant Bacillus subtilis Expressing HA1 Protein of H9N2 Avian Influenza Virus. Curr. Microbiol., 81.","DOI":"10.1007\/s00284-023-03548-x"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e02090-24","DOI":"10.1128\/jvi.02090-24","article-title":"Identification and characterization of a broadly neutralizing and protective nanobody against the HA1 domain of H5 avian influenza virus hemagglutinin","volume":"99","author":"Xu","year":"2025","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2275","DOI":"10.1007\/s007050170002","article-title":"Universal primer set for the full-length amplification of all influenza A viruses","volume":"146","author":"Hoffmann","year":"2001","journal-title":"Arch. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Vasquez, R., Bagon, B.B., Song, J.H., Han, N.S., and Kang, D.-K. (2022). A novel, non-GMO surface display in Limosilactobacillus fermentum mediated by cell surface hydrolase without anchor motif. BMC Microbiol., 22.","DOI":"10.1186\/s12866-022-02608-9"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6633","DOI":"10.1128\/AEM.71.11.6633-6643.2005","article-title":"Detection, characterization, and in vitro and in vivo expression of genes encoding S-proteins in Lactobacillus gallinarum strains isolated from chicken crops","volume":"71","author":"Hagen","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2410","DOI":"10.1128\/AEM.01752-09","article-title":"Characterization of a novel LysM domain from Lactobacillus fermentum bacteriophage endolysin and its use as an anchor to display heterologous proteins on the surfaces of lactic acid bacteria","volume":"76","author":"Hu","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"880","DOI":"10.1128\/AEM.72.1.880-889.2006","article-title":"Novel surface display system for proteins on non-genetically modified gram-positive bacteria","volume":"72","author":"Bosma","year":"2006","journal-title":"Appl. Environ. Microb."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5943","DOI":"10.1128\/AEM.68.12.5943-5951.2002","article-title":"Surface Display of Foreign Epitopes on the Lactobacillus brevis S-Layer","volume":"68","author":"Kahala","year":"2002","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Mathiesen, G., \u00d8verland, L., Kuczkowska, K., and Eijsink, V.G.H. (2020). Anchoring of heterologous proteins in multiple Lactobacillus species using anchors derived from Lactobacillus plantarum. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-66531-7"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1769","DOI":"10.1016\/S0264-410X(02)00027-0","article-title":"Comparison of the immune responses induced by local immunizations with recombinant Lactobacillus plantarum producing tetanus toxin fragment C in different cellular locations","volume":"20","author":"Reveneau","year":"2002","journal-title":"Vaccine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/S0958-1669(99)00018-X","article-title":"Functionality of probiotics and intestinal lactobacilli: Light in the intestinal tract tunnel","volume":"10","author":"Vaughan","year":"1999","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1111\/j.1574-6968.1984.tb01246.x","article-title":"Host specificity of the colonization of murine gastric epithelium by lactobacilli","volume":"24","author":"Lin","year":"1984","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ravikumar, R., Chan, J., and Prabakaran, M. (2022). Vaccines against Major Poultry Viral Diseases: Strategies to Improve the Breadth and Protective Efficacy. Viruses, 14.","DOI":"10.3390\/v14061195"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e00061-18","DOI":"10.1128\/mSphere.00061-18","article-title":"Lactobacillus Mucosal Vaccine Vectors: Immune Responses against Bacterial and Viral Antigens","volume":"3","author":"LeCureux","year":"2018","journal-title":"mSphere"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/7\/1649\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:08:51Z","timestamp":1760033331000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/7\/1649"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,11]]},"references-count":37,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["microorganisms13071649"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms13071649","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,11]]}}}