{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T09:18:15Z","timestamp":1777540695057,"version":"3.51.4"},"reference-count":42,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T00:00:00Z","timestamp":1606694400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004663","name":"Ministry of Science and Technology, Taiwan","doi-asserted-by":"publisher","award":["109-2313-B-007-001"],"award-info":[{"award-number":["109-2313-B-007-001"]}],"id":[{"id":"10.13039\/501100004663","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.<\/jats:p>","DOI":"10.3390\/vaccines8040710","type":"journal-article","created":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T20:10:22Z","timestamp":1606767022000},"page":"710","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses"],"prefix":"10.3390","volume":"8","author":[{"given":"Neos","family":"Tang","sequence":"first","affiliation":[{"name":"Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5240-2808","authenticated-orcid":false,"given":"Chun-Yi","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, National Taiwan University Children Hospital, Taipei 100226, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2169-0157","authenticated-orcid":false,"given":"Shih-Che","family":"Sue","sequence":"additional","affiliation":[{"name":"Department of Life Science, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ting-Hsuan","family":"Chen","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jia-Tsrong","family":"Jan","sequence":"additional","affiliation":[{"name":"Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9670-3821","authenticated-orcid":false,"given":"Ming-Hsi","family":"Huang","sequence":"additional","affiliation":[{"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan 35053, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2295-6412","authenticated-orcid":false,"given":"Chung-Hsiung","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Food Science, National Taiwan Ocean University, Keelung 202301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4415-7811","authenticated-orcid":false,"given":"Chung-Chu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, MacKay Memorial Hospital, Hsinchu 30013, Taiwan"},{"name":"Teaching Center of Natural Science, Minghsin University of Science and Technology, Hsinchu 202301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bor-Luen","family":"Chiang","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, National Taiwan University Children Hospital, Taipei 100226, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9291-260X","authenticated-orcid":false,"given":"Li-Min","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, National Taiwan University Children Hospital, Taipei 100226, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1672-5255","authenticated-orcid":false,"given":"Suh-Chin","family":"Wu","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan"},{"name":"Department of Medical Science, National Tsing Hua University, Hsinchu 30013, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,11,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1099\/vir.0.83419-0","article-title":"H5N1 avian influenza re-emergence of Lake Qinghai: Phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation","volume":"89","author":"Wang","year":"2008","journal-title":"J. Gen. Virol."},{"key":"ref_2","first-page":"931","article-title":"Emergence and pandemic potential of swine-origin H1N1 influenza virus","volume":"459","author":"Neumann","year":"2009","journal-title":"Nat. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"792","DOI":"10.4161\/hv.6.10.12915","article-title":"Pre-pandemic and pandemic influenza vaccines","volume":"6","author":"Rockman","year":"2010","journal-title":"Hum. Vaccines"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.tim.2011.10.003","article-title":"The changing nature of avian influenza A virus (H5N1)","volume":"20","author":"Watanabe","year":"2012","journal-title":"Trends Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1056\/NEJMoa040419","article-title":"World Health Organization International Avian Influenza Investigative Team. Avian influenza A (H5N1) in 10 patients in Vietnam","volume":"350","author":"Tran","year":"2004","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1038\/nm1477","article-title":"Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia","volume":"12","author":"Simmons","year":"2006","journal-title":"Nat. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1534","DOI":"10.1126\/science.1213362","article-title":"Airborne Transmission of Influenza A\/H5N1 Virus Between Ferrets","volume":"336","author":"Herfst","year":"2012","journal-title":"Science"},{"key":"ref_8","first-page":"420","article-title":"Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA\/H1N1 virus in ferrets","volume":"486","author":"Imai","year":"2012","journal-title":"Nat. Cell Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.ejpb.2015.05.023","article-title":"Current and next generation influenza vaccines: Formulation and production strategies","volume":"94","author":"Soema","year":"2015","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1038\/mi.2010.55","article-title":"Topical immunization strategies","volume":"3","author":"Czerkinsky","year":"2010","journal-title":"Mucosal Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/S0169-409X(01)00162-4","article-title":"Nasal vaccines","volume":"51","author":"Davis","year":"2001","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1038\/nri1439","article-title":"NALT-versus Peyer\u2019s-patch-mediated mucosal immunity","volume":"4","author":"Kiyono","year":"2004","journal-title":"Nat. Rev. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1146\/annurev-bioeng-071811-150054","article-title":"Mucosal Vaccine Design and Delivery","volume":"14","author":"Woodrow","year":"2012","journal-title":"Annu. Rev. Biomed. Eng."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1016\/j.jconrel.2012.02.006","article-title":"Crossing the barrier: Targeting epithelial receptors for enhanced oral vaccine delivery","volume":"160","author":"Devriendt","year":"2012","journal-title":"J. Control. Release"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4302","DOI":"10.1016\/j.vaccine.2009.05.027","article-title":"In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli","volume":"27","author":"Liang","year":"2009","journal-title":"Vaccine"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"449","DOI":"10.3109\/08820130903563998","article-title":"Heat-Labile Enterotoxins as Adjuvants or Anti-Inflammatory Agents","volume":"39","author":"Liang","year":"2010","journal-title":"Immunol. Investig."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Lin, S.-C., Huang, M.-H., Tsou, P.-C., Huang, L.-M., Chong, P., and Wu, S.-C. (2011). Recombinant Trimeric HA Protein Immunogenicity of H5N1 Avian Influenza Viruses and Their Combined Use with Inactivated or Adenovirus Vaccines. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0020052"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12011","DOI":"10.1128\/JVI.01316-10","article-title":"Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses","volume":"84","author":"Liu","year":"2010","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Huang, M.-H., Lin, S.-C., Hsiao, C.-H., Chao, H.-J., Yang, H.-R., Liao, C.-C., Chuang, P.-W., Wu, H.-P., Huang, C.-Y., and Leng, C.-H. (2010). Emulsified Nanoparticles Containing Inactivated Influenza Virus and CpG Oligodeoxynucleotides Critically Influences the Host Immune Responses in Mice. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0012279"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.jcv.2007.02.005","article-title":"Hemagglutinin pseudotyped lentiviral particles: Characterization of a new method for avian H5N1 influenza sero-diagnosis","volume":"39","author":"Nefkens","year":"2007","journal-title":"J. Clin. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Lin, S.-C., Liu, W.-C., Jan, J.-T., and Wu, S.-C. (2014). Glycan Masking of Hemagglutinin for Adenovirus Vector and Recombinant Protein Immunizations Elicits Broadly Neutralizing Antibodies against H5N1 Avian Influenza Viruses. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0092822"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1804","DOI":"10.1016\/j.vaccine.2004.11.010","article-title":"Mucosal adjuvants","volume":"23","author":"Freytag","year":"2005","journal-title":"Vaccine"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1038\/ni.2567","article-title":"High-affinity IgA needs TH17 cell functional plasticity","volume":"14","author":"Milpied","year":"2013","journal-title":"Nat. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"14292","DOI":"10.1073\/pnas.1009234107","article-title":"Proinflammatory T helper type 17 cells are effective B-cell helpers","volume":"107","author":"Mitsdoerffer","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4321","DOI":"10.1016\/j.vaccine.2015.03.082","article-title":"Use of recombinant flagellin in oil-in-water emulsions enhances hemagglutinin-specific mucosal IgA production and IL-17 secreting T cells against H5N1 avian influenza virus infection","volume":"33","author":"Lai","year":"2015","journal-title":"Vaccine"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"9026","DOI":"10.1016\/j.vaccine.2011.09.033","article-title":"Development and characterization of chitosan coated poly-(\u025b-caprolactone) nanoparticulate system for effective immunization against influenza","volume":"29","author":"Gupta","year":"2011","journal-title":"Vaccine"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.vetmic.2012.05.035","article-title":"Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate\/chitosan nanoparticles induces protection against highly pathogenic influenza A virus","volume":"160","author":"Moon","year":"2012","journal-title":"Vet. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Meng, S., Liu, Z., Xu, L., Li, L., Mei, S., Bao, L., Deng, W., Li, L., Lei, R., and Xie, L. (2011). Intranasal immunization with recombinant HA and mast cell activator C48\/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0019863"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.it.2012.01.003","article-title":"Innate IL-17 and IL-22 responses to enteric bacterial pathogens","volume":"33","author":"Rubino","year":"2012","journal-title":"Trends Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1221","DOI":"10.1002\/eji.200838742","article-title":"TLR2-activated human langerhans cells promote Th17 polarization via IL-1beta, TGF-beta and IL-23","volume":"39","author":"Aliahmadi","year":"2009","journal-title":"Eur. J. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4103","DOI":"10.4049\/jimmunol.1301970","article-title":"Periodontal Pathogens Directly Promote Autoimmune Experimental Arthritis by Inducing a TLR2- and IL-1\u2013Driven Th17 Response","volume":"192","author":"Koenders","year":"2014","journal-title":"J. Immunol."},{"key":"ref_32","first-page":"644","article-title":"Toll-like Receptor 2-and 4-Mediated Reciprocal Th17 and Antibody Responses to Group A Streptococcus Infection","volume":"215","author":"Wang","year":"2016","journal-title":"J. Infect. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3469","DOI":"10.4049\/jimmunol.0801814","article-title":"Tc17, a Unique Subset of CD8 T Cells That Can Protect against Lethal Influenza Challenge","volume":"182","author":"Hamada","year":"2009","journal-title":"J. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"7353","DOI":"10.4049\/jimmunol.0900657","article-title":"IL-10 Deficiency Unleashes an Influenza-Specific Th17 Response and Enhances Survival against High-Dose Challenge","volume":"182","author":"McKinstry","year":"2009","journal-title":"J. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"5301","DOI":"10.4049\/jimmunol.0900995","article-title":"Critical Role of IL-17RA in Immunopathology of Influenza Infection","volume":"183","author":"Crowe","year":"2009","journal-title":"J. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Maroof, A., Yorgensen, Y.M., Li, Y., and Evans, J.T. (2014). Intranasal Vaccination Promotes Detrimental Th17-Mediated Immunity against Influenza Infection. PLoS Pathog., 10.","DOI":"10.1371\/journal.ppat.1003875"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/j.coi.2013.03.011","article-title":"Th17 cell based vaccines in mucosal immunity","volume":"25","author":"Kumar","year":"2013","journal-title":"Curr. Opin. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1146\/annurev.immunol.021908.132710","article-title":"IL-17 and Th17 Cells","volume":"27","author":"Korn","year":"2009","journal-title":"Annu. Rev. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Wang, X., Ma, K., Chen, M., Ko, K.-H., Zheng, B.-J., and Lu, L. (2016). IL-17A Promotes Pulmonary B-1a Cell Differentiation via Induction of Blimp-1 Expression during Influenza Virus Infection. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005367"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"972","DOI":"10.1038\/mi.2012.135","article-title":"Interleukin-17-dependent CXCL13 mediates mucosal vaccine\u2013induced immunity against tuberculosis","volume":"6","author":"Gopal","year":"2013","journal-title":"Mucosal Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"896","DOI":"10.1056\/NEJMoa030595","article-title":"Use of the inactivated intranasal influenza vaccine and the risk of Bell\u2019s palsy in Switzerland","volume":"350","author":"Mutsch","year":"2004","journal-title":"N. Engl. J. Med."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Lewis, D.J., Huo, Z., Barnett, S., Kromann, I., Giemza, R., Galiza, E., Woodrow, M., Thierry-Carstensen, B., Andersen, P., and Novicki, D. (2009). Transient facial nerve paralysis (Bell\u2019s palsy) following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat labile toxin. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0006999"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/8\/4\/710\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:39:48Z","timestamp":1760179188000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/8\/4\/710"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,11,30]]},"references-count":42,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2020,12]]}},"alternative-id":["vaccines8040710"],"URL":"https:\/\/doi.org\/10.3390\/vaccines8040710","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,11,30]]}}}