title: Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomerization, hemagglutination, and cross-protective immunity in ferrets.
abstract: The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. We have developed bacterial systems for expression and purification of properly folded functional hemagglutinin as a rapid response to emerging pandemic strains. A recombinant H5N1 (A/Vietnam/1203/2004) hemagglutinin globular domain (HA1) was produced in Escherichia coli under controlled redox refolding conditions. Importantly, the properly folded HA1(1-320), i.e., HA1 lacking amino acids 321 to 330, contained ≥75% functional oligomers without addition of foreign oligomerization sequence. Site-directed mutagenesis mapped the oligomerization signal to the HA1 N-terminal Ile-Cys-Ile residues at positions 3 to 5. The purified HA1 oligomers (but not monomers) bound fetuin and agglutinated red blood cells. Upon immunization of rabbits, the oligomeric HA1(1-320) elicited potent neutralizing antibodies against homologous and heterologous H5N1 viruses more rapidly than HA1(28-320) containing only monomers. Ferrets vaccinated with oligomeric HA1 (but not monomeric HA1 with the N terminus deleted) at 15 and 3 μg/dose were fully protected from lethality and weight loss after challenge with homologous H5N1 (A/Vietnam/1203/2004, clade 1) virus, as well as heterologous clade 2.2 H5N1 (A/WooperSwan/Mongolia/244/2005) virus. Protection was associated with a significant reduction in viral loads in the nasal washes of homologous and heterologous virus challenged ferrets. This is the first study that describes the presence of an N-terminal oligomerization sequence in the globular domain of influenza virus hemagglutinin. Our findings suggest that functional oligomeric rHA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.
authors.affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: Surender
authors.initials: S
authors.lastname: Khurana
authors.forename: Swati
authors.initials: S
authors.lastname: Verma
authors.forename: Nitin
authors.initials: N
authors.lastname: Verma
authors.forename: Corey J
authors.initials: CJ
authors.lastname: Crevar
authors.forename: Donald M
authors.initials: DM
authors.lastname: Carter
authors.forename: Jody
authors.initials: J
authors.lastname: Manischewitz
authors.forename: Lisa R
authors.initials: LR
authors.lastname: King
authors.forename: Ted M
authors.initials: TM
authors.lastname: Ross
authors.forename: Hana
authors.initials: H
authors.lastname: Golding
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005289:Ferrets; D005822:Genetic Vectors; D006384:Hemagglutination; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology*; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D009297:Nasal Mucosa / Q000821:virology; D009976:Orthomyxoviridae Infections / Q000401:mortality / Q000517:prevention & control; D011485:Protein Binding; D055503:Protein Multimerization; D011817:Rabbits; D016019:Survival Analysis; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D019562:Viral Load; D000509:alpha-Fetoproteins / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D014614:Vaccines, Synthetic; D000509:alpha-Fetoproteins