{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T23:06:45Z","timestamp":1776121605344,"version":"3.50.1"},"reference-count":43,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,11,27]],"date-time":"2021-11-27T00:00:00Z","timestamp":1637971200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing foreign antigens. In this study, we generated and evaluated the live mucosal recombinant vaccine by integrating genes encoding influenza virus neuraminidase (NA) of the N2 subtype into the DNA of the probiotic strain Enterococcus faecium L3 (L3). We confirmed NA expression in the pili of L3 using immune electron microscopy. Mice were fed with a probiotic vaccine containing the NA gene (L3-NA) or pure L3. Oral administration of L3-NA caused detectable increase in virus-specific serum IgG and local IgA after the third feeding. Immunization with L3-NA increased the survival rate by 34% when the mice were infected using A(H1N1)pdm09 influenza virus after the third feeding. After S. pneumoniae post-influenza infection, the L3-NA-immunized mice were 50% more protected from lethality in comparison with L3-fed mice. Thus, a live probiotic vaccine candidate based on L3 induced the formation of systemic and local immunity and provide partial protection against complicated influenza.<\/jats:p>","DOI":"10.3390\/microorganisms9122446","type":"journal-article","created":{"date-parts":[[2021,11,30]],"date-time":"2021-11-30T05:42:10Z","timestamp":1638250930000},"page":"2446","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9794-3520","authenticated-orcid":false,"given":"Yulia","family":"Desheva","sequence":"first","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Galina","family":"Leontieva","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tatiana","family":"Kramskaya","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4824-3621","authenticated-orcid":false,"given":"Tatiana","family":"Gupalova","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5245-7315","authenticated-orcid":false,"given":"Igor","family":"Losev","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eugenia","family":"Kuleshevich","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Bormotova","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olga","family":"Kopteva","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Polina","family":"Kudar","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander","family":"Suvorov","sequence":"additional","affiliation":[{"name":"Scientific and Educational Center \u201cMolecular Bases of Interaction of Microorganisms and Human\u201d of the World-Class Research Center \u201cCenter for Personalized Medicine\u201d FSBSI \u201cIEM\u201d, 197376 Saint Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1099\/mic.0.049932-0","article-title":"Construction of Bifidobacterium infantis as a live oral vaccine that expresses antigens of the major fimbrial subunit (CfaB) and the B subunit of heat-labile enterotoxin (LTB) from enterotoxigenic Escherichia coli","volume":"158","author":"Ma","year":"2012","journal-title":"Microbiology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.virol.2014.12.017","article-title":"Lactococcus lactis displayed neuraminidase confers cross protective immunity against influenza A viruses in mice","volume":"476","author":"Lei","year":"2015","journal-title":"Virology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1136\/archdischild-2016-312165","article-title":"Live attenuated influenza vaccine (LAIV): Recent effectiveness results from the USA and implications for LAIV programmes elsewhere","volume":"103","author":"Pebody","year":"2017","journal-title":"Arch. Dis. Child."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e20182094","DOI":"10.1542\/peds.2018-2094","article-title":"Live Attenuated and Inactivated Influenza Vaccine Effectiveness","volume":"143","author":"Chung","year":"2019","journal-title":"Pediatrics"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1007\/s12602-018-9457-0","article-title":"Anti\u2013Influenza Activity of Enterocin B In vitro and Protective Effect of Bacteriocinogenic Enterococcal Probiotic Strain on Influenza Infection in Mouse Model","volume":"11","author":"Ermolenko","year":"2018","journal-title":"Probiotics Antimicrob. Proteins"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/j.ics.2005.11.030","article-title":"Antagonistic activity of Enterococcus faecium L3 against different groups of pathogenic streptococci","volume":"1289","author":"Yermolenko","year":"2006","journal-title":"Int. Congr. Ser."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"265","DOI":"10.3920\/BM2010.0008","article-title":"The influence of probiotic Enterococcus faecium strain L5 on the microbiota and cytokines expression in rats with dysbiosis induced by antibiotics","volume":"1","author":"Tarasova","year":"2010","journal-title":"Benef. Microbes"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e01622-15","DOI":"10.1128\/genomeA.01622-15","article-title":"Draft Genome Sequence of Probiotic Enterococcus faecium Strain L-3","volume":"4","author":"Karaseva","year":"2016","journal-title":"Genome Announc."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1038\/nrmicro1443","article-title":"Pili in Gram-positive pathogens","volume":"4","author":"Telford","year":"2006","journal-title":"Nat. Rev. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1016\/j.resmic.2012.10.012","article-title":"Pili of Gram-positive bacteria: Roles in host colonization","volume":"163","author":"Danne","year":"2012","journal-title":"Res. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6588","DOI":"10.1016\/j.vaccine.2006.05.039","article-title":"Cross-protective immunity in mice induced by live-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1) viruses","volume":"24","author":"Lu","year":"2006","journal-title":"Vaccine"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e00307-19","DOI":"10.1128\/mBio.00307-19","article-title":"Antigenic drift of the influenza A (H1N1) pdm09 virus neuraminidase results in reduced effectiveness of A\/California\/7\/2009 (H1N1pdm09)-specific antibodies","volume":"10","author":"Gao","year":"2019","journal-title":"Mbio"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2465","DOI":"10.3390\/v6062465","article-title":"In the shadow of hemagglutinin: A growing interest in influenza viral neuraminidase and its role as a vaccine antigen","volume":"6","author":"Wohlbold","year":"2014","journal-title":"Viruses"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Giurgea, L.T., Morens, D.M., Taubenberger, J.K., and Memoli, M.J. (2020). Influenza Neuraminidase: A Neglected Protein and Its Potential for a Better Influenza Vaccine. Vaccines, 8.","DOI":"10.3390\/vaccines8030409"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"S75","DOI":"10.1093\/infdis\/jiz017","article-title":"Neuraminidase, the Forgotten Surface Antigen, Emerges as an Influenza Vaccine Target for Broadened Protection","volume":"219","author":"Eichelberger","year":"2019","journal-title":"J. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e02332-17","DOI":"10.1128\/mBio.02332-17","article-title":"NAction! How Can Neuraminidase-Based Immunity Contribute to Better Influenza Virus Vaccines?","volume":"9","author":"Krammer","year":"2018","journal-title":"MBio"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.coi.2018.03.025","article-title":"Neuraminidase as an influenza vaccine antigen: A low hanging fruit, ready for picking to improve vaccine effectiveness","volume":"53","author":"Eichelberger","year":"2018","journal-title":"Curr. Opin. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1016\/j.virol.2012.05.006","article-title":"Influenza M1 VLPs containing neuraminidase induce heterosubtypic cross-protection","volume":"430","author":"Quan","year":"2012","journal-title":"Virology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1111\/tbed.12327","article-title":"A Systematic Review of the Comparative Epidemiology of Avian and Human Influenza A H5N1 and H7N9\u2014Lessons and Unanswered Questions","volume":"63","author":"Bui","year":"2015","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/S0968-0004(00)01561-9","article-title":"Cn3D: Sequence and structure views for Entrez","volume":"25","author":"Wang","year":"2000","journal-title":"Trends Biochem. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1007\/s00726-006-0485-9","article-title":"Prediction of linear B-cell epitopes using amino acid pair antigenicity scale","volume":"33","author":"Chen","year":"2007","journal-title":"Amino Acids"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1002\/prot.21078","article-title":"Prediction of continuous B-cell epitopes in an antigen using recurrent neural network","volume":"65","author":"Saha","year":"2006","journal-title":"Proteins: Struct. Funct. Bioinform."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1186\/1745-7580-2-2","article-title":"Improved method for predicting linear B-cell epitopes","volume":"2","author":"Larsen","year":"2006","journal-title":"Immunome Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1093\/protein\/gzn075","article-title":"COBEpro: A novel system for predicting continuous B-cell epitopes","volume":"22","author":"Sweredoski","year":"2008","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Ponomarenko, J.V., 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_26","doi-asserted-by":"crossref","unstructured":"Rubinstein, N.D., Mayrose, I., Martz, E., and Pupko, T. (2009). Epitopia: A web-server for predicting B-cell epitopes. BMC Bioinform., 10.","DOI":"10.1186\/1471-2105-10-287"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Gupalova, T., Leontieva, G., Kramskaya, T., Grabovskaya, K., Kuleshevich, E., and Suvorov, A. (2019). Development of experimental pneumococcal vaccine for mucosal immunization. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0218679"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Waterborg, J.H. (2009). The Lowry method for protein quantitation. The Protein Protocols Handbook, Humana Press.","DOI":"10.1007\/978-1-59745-198-7_2"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Desheva, Y., Smolonogina, T., Donina, S., and Rudenko, L. (2020). Study of Neuraminidase-Inhibiting Antibodies in Clinical Trials of Live Influenza Vaccines. Antibodies, 9.","DOI":"10.3390\/antib9020020"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Silver, L.M. (1995). Mouse Genetics: Concepts and Applications, Oxford University Press.","DOI":"10.1093\/oso\/9780195075540.001.0001"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1278","DOI":"10.1016\/j.vaccine.2010.11.080","article-title":"Linear B-cell epitope mapping of neuraminidases of the 2009 A H1N1 viruses based on immunoinformatics","volume":"29","author":"Huang","year":"2011","journal-title":"Vaccine"},{"key":"ref_32","first-page":"551","article-title":"Sialidases in biological systems","volume":"65","author":"Melzig","year":"2010","journal-title":"Die Pharm.-Int. J. Pharm. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1086\/381547","article-title":"Nasopharyngeal carriage of Streptococcus pneumoniae by adults and children in community and family settings","volume":"38","author":"Raz","year":"2004","journal-title":"Clin. Infect. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1016\/j.antiviral.2013.09.018","article-title":"Universal anti-neuraminidase antibody inhibiting all influenza A subtypes","volume":"100","author":"Doyle","year":"2013","journal-title":"Antivir. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.jaut.2014.07.005","article-title":"Epitope-based approaches to a universal influenza vaccine","volume":"54","author":"Gottlieb","year":"2014","journal-title":"J. Autoimmun."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.virol.2017.11.018","article-title":"A novel peptide-based vaccine candidate with protective efficacy against influenza A in a mouse model","volume":"515","author":"Meijerhof","year":"2018","journal-title":"Virology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"525","DOI":"10.3389\/fimmu.2018.00525","article-title":"Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes","volume":"9","author":"Soema","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_38","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_39","doi-asserted-by":"crossref","unstructured":"De Azevedo, M., Karczewski, J., Lef\u00e9vre, F., Azevedo, V., Miyoshi, A., Wells, J.M., Langella, P., and Chatel, J.-M. (2012). In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A. BMC Microbiol., 12.","DOI":"10.1186\/1471-2180-12-299"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Gupalova, T., Leontieva, G., Kramskaya, T., Grabovskaya, K., Bormotova, E., Korjevski, D., and Suvorov, A. (2018). Development of experimental GBS vaccine for mucosal immunization. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0196564"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.virol.2017.06.006","article-title":"Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets","volume":"509","author":"Smith","year":"2017","journal-title":"Virology"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Kramskaya, T., Leontieva, G., Desheva, Y., Grabovskaya, K., Gupalova, T., Rudenko, L., and Suvorov, A. (2019). Combined immunization with attenuated live influenza vaccine and chimeric pneumococcal recombinant protein improves the outcome of virus-bacterial infection in mice. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0222148"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Taghinezhad, S.S., Mohseni, A.H., Berm\u00fadez-Humar\u00e1n, L.G., Casolaro, V., Cortes-Perez, N.G., Keyvani, H., and Simal-Gandara, J. (2021). Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease. Vaccines, 9.","DOI":"10.3390\/vaccines9050466"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/9\/12\/2446\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:36:44Z","timestamp":1760168204000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/9\/12\/2446"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,27]]},"references-count":43,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["microorganisms9122446"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms9122446","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,27]]}}}