title: Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion.
abstract: Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design.
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Gong
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: He
authors.initials: H
authors.lastname: Yin
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Shi
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Guan
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Xiaoqiu
authors.initials: X
authors.lastname: He
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Yongjiao
authors.initials: Y
authors.lastname: Yu
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Kuai
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China; Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Chunlai
authors.initials: C
authors.lastname: Jiang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China; Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.affiliation: State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun, Jilin, China.
authors.forename: Song
authors.initials: S
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China; Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, Changchun, Jilin, China. Electronic address: shanym@jlu.edu.cn.
authors.forename: Yaming
authors.initials: Y
authors.lastname: Shan
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques; D003198:Computer Simulation; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000276:immunology*; D007252:Influenza Vaccines / Q000276:immunology*; D007251:Influenza, Human / Q000276:immunology*; D008561:Membrane Fusion; D051379:Mice; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000276:immunology*; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines
keywords: Conformational change; Hemagglutinin stem; Influenza virus; Membrane fusion; Neutralizing activities