{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T21:40:12Z","timestamp":1761946812541,"version":"build-2065373602"},"reference-count":23,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2017,12,23]],"date-time":"2017-12-23T00:00:00Z","timestamp":1513987200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The highly pathogenic avian influenza (HPAI) H5N1 virus remains to be one of the world\u2019s largest pandemic threats due to the emergence of new variants. The rapid evolution of new sub-lineages is currently the greatest challenge in vaccine development. In this study, we developed an epitope modified non-pathogenic H5N3 (A\/duck\/Singapore\/97) vaccine for broad protection against influenza H5 subtype. H5N3 hemagglutinin (HA) mutant reassortant viruses with A\/Puerto Rico\/8\/34 (PR8) backbone were generated by mutating amino acids at the 140th loop and 190th \u03b1-helix of hemagglutinin. The cross-neutralizing efficacy of reverse genetics-derived H5N3HA (RG-H5N3HA) mutants was confirmed by testing reactivity with reference chicken anti-H5N1 clade 2 virus sera. Furthermore, RG-H5N3HA mutant immunized mice induced cross-neutralizing antibodies and cross-protection against distinct H5N1 viral infection. Our findings suggest that the use of non-pathogenic H5 viruses antigenically related to HPAI-H5N1 allows for the development of broadly protective vaccines and reduces the need for biosafety level 3 (BSL3) containment facilities.<\/jats:p>","DOI":"10.3390\/v10010002","type":"journal-article","created":{"date-parts":[[2017,12,26]],"date-time":"2017-12-26T03:06:38Z","timestamp":1514257598000},"page":"2","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Broadening the H5N3 Vaccine Immunogenicity against H5N1 Virus by Modification of Neutralizing Epitopes"],"prefix":"10.3390","volume":"10","author":[{"given":"Subaschandrabose","family":"Rajesh Kumar","sequence":"first","affiliation":[{"name":"Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sharenya","family":"Chelvaretnam","sequence":"additional","affiliation":[{"name":"Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yunrui","family":"Tan","sequence":"additional","affiliation":[{"name":"Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mookkan","family":"Prabakaran","sequence":"additional","affiliation":[{"name":"Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2017,12,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"842","DOI":"10.3201\/eid2105.141927","article-title":"Rapid Emergence of Highly Pathogenic Avian Influenza Subtypes from a Subtype H5N1 Hemagglutinin Variant","volume":"21","author":"Guo","year":"2015","journal-title":"Emerg. Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1128\/CMR.00097-12","article-title":"Traditional and new influenza vaccines","volume":"26","author":"Wong","year":"2013","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Prabakaran, M., Leyrer, S., He, F., Auer, S., Kumar, S.R., Kindsmueller, K., Mytle, N., Schneider, J., Lockhart, S., and Kwang, J. (2014). Progress toward a Universal H5N1 Vaccine: A Recombinant Modified Vaccinia Virus Ankara-Expressing Trivalent Hemagglutinin Vaccine. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0107316"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Yang, P., Duan, Y., Zhang, P., Li, Z., Wang, C., Dong, M., Tang, C., Xing, L., Gu, H., and Zhao, Z. (2012). Multiple-Clade H5N1 Influenza Split Vaccine Elicits Broad Cross Protection against Lethal Influenza Virus Challenge in Mice by Intranasal Vaccination. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0030252"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1073\/pnas.1012457108","article-title":"Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development","volume":"108","author":"Ducatez","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"13538","DOI":"10.1073\/pnas.0806901105","article-title":"A consensus hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses","volume":"105","author":"Chen","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"11822","DOI":"10.1128\/JVI.00891-10","article-title":"Neutralizing epitopes of influenza virus hemagglutinin: Target for the development of a universal vaccine against H5N1 lineages","volume":"84","author":"Prabakaran","year":"2010","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1086\/428948","article-title":"Cross-reactivity to highly pathogenic avian influenza H5N1 viruses after vaccination with nonadjuvanted and MF59-adjuvanted influenza A\/duck\/Singapore\/97 (H5N3) vaccine: A potential priming strategy","volume":"191","author":"Stephenson","year":"2005","journal-title":"J. Infect. Dis."},{"key":"ref_9","unstructured":"WHO (2012). Antigenic and genetic characteristics of zoonotic influenza viruses and development of candidate vaccine viruses for pandemic preparedness. Wkly. Epidemiol. Rec., 87, 97\u2013108."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"9346","DOI":"10.1073\/pnas.1502762112","article-title":"Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites","volume":"112","author":"Winarski","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Prabakaran, M., Ho, H.T., Prabhu, N., Velumani, S., Szyporta, M., He, F., Chan, K.P., Chen, L.M., Matsuoka, Y., and Donis, R.O. (2009). Development of Epitope-Blocking ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0004566"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"505","DOI":"10.2307\/1591214","article-title":"Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum","volume":"35","author":"King","year":"1991","journal-title":"Avian Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2497","DOI":"10.1099\/0022-1317-83-10-2497","article-title":"Structure of antigenic sites on the hemagglutinin molecule of H5 influenza virus and phenotypic variation of escape mutants","volume":"83","author":"Kaverin","year":"2002","journal-title":"J. Gen. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3201","DOI":"10.1128\/JVI.02175-09","article-title":"Gastrointestinal delivery of baculovirus displaying influenza virus hemagglutinin protects mice against heterologous H5N1 infection","volume":"84","author":"Prabakaran","year":"2010","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4079","DOI":"10.1016\/j.vaccine.2010.03.078","article-title":"The impact of key amino acid substitutions in the hemagglutinin of influenza A (H3N2) viruses on vaccine production and antibody response","volume":"28","author":"Zhongying","year":"2010","journal-title":"Vaccine"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Suguitan, A.L., McAuliffe, J., Milis, K.L., Jin, H., Duke, G., Lu, B., Luke, C.J., Murphy, B., Swayne, D.E., and Kemble, G. (2006). Live, attenuated influenza A H5N1 candidate vaccines provide broad cross protection in mice and ferrets. PLoS Med., 3.","DOI":"10.1371\/journal.pmed.0030360"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1016\/0264-410X(91)90055-B","article-title":"Efficacy of nucleoprotein and hemagglutinin antigens expressed in fowl pox virus as vaccine for influenza in chickens","volume":"9","author":"Webster","year":"1991","journal-title":"Vaccine"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/j.virol.2012.09.016","article-title":"Antigenic characterization of recent H5N1 highly pathogenic avian influenza viruses circulating in Egyptian poultry","volume":"435","author":"Beato","year":"2013","journal-title":"Virology"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Shore, D.A., Yang, H., Balish, A.L., Shepard, S.S., Carney, P.J., Chang, J.C., Davis, C.T., Donis, R.O., Villanueva, J.M., and Klimov, A.I. (2013). Structural and Antigenic Variation among Diverse Clade 2 H5N1 Viruses. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0075209"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1146\/annurev.biochem.69.1.531","article-title":"Receptor binding and membrane fusion in virus entry: The influenza hemagglutinin","volume":"69","author":"Skehel","year":"2000","journal-title":"Annu. Rev. Biochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1362","DOI":"10.1086\/379045","article-title":"Cross-reactive H1N1 antibody responses to a live attenuated influenza vaccine in children: Implication for selection of vaccine strains","volume":"188","author":"Lee","year":"2003","journal-title":"J. Infect. Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e01070-14","DOI":"10.1128\/mBio.01070-14","article-title":"Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site","volume":"5","author":"Koel","year":"2014","journal-title":"mBio"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1382","DOI":"10.1016\/j.jmb.2008.04.016","article-title":"Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity","volume":"381","author":"Stevens","year":"2008","journal-title":"J. Mol. Biol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/10\/1\/2\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:55:17Z","timestamp":1760208917000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/10\/1\/2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,12,23]]},"references-count":23,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2018,1]]}},"alternative-id":["v10010002"],"URL":"https:\/\/doi.org\/10.3390\/v10010002","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2017,12,23]]}}}