title: Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge.
abstract: Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Gayathri
authors.initials: G
authors.lastname: Bommakanti
authors.forename: Michael P
authors.initials: MP
authors.lastname: Citron
authors.forename: Robert W
authors.initials: RW
authors.lastname: Hepler
authors.forename: Cheryl
authors.initials: C
authors.lastname: Callahan
authors.forename: Gwendolyn J
authors.initials: GJ
authors.lastname: Heidecker
authors.forename: Tariq Ahmad
authors.initials: TA
authors.lastname: Najar
authors.forename: Xianghan
authors.initials: X
authors.lastname: Lu
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Joyce
authors.forename: John W
authors.initials: JW
authors.lastname: Shiver
authors.forename: Danilo R
authors.initials: DR
authors.lastname: Casimiro
authors.forename: Jan
authors.initials: J
authors.lastname: ter Meulen
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Liang
authors.forename: Raghavan
authors.initials: R
authors.lastname: Varadarajan
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000276:immunology*; D007252:Influenza Vaccines / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009154:Mutation; D009976:Orthomyxoviridae Infections / Q000276:immunology* / Q000517:prevention & control*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines