title: Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines.
abstract: The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.
authors.affiliation: Departments of Chemical Engineering and Bioengineering, Stanford University, Stanford, CA 94305.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lu
authors.forename: John P
authors.initials: JP
authors.lastname: Welsh
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D057134:Antibodies, Neutralizing / Q000737:chemistry; D000914:Antibodies, Viral / Q000737:chemistry; D000956:Antigens, Viral / Q000096:biosynthesis; D017122:Bacteriophage T4 / Q000737:chemistry; D002474:Cell-Free System; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry*; D007252:Influenza Vaccines / Q000096:biosynthesis*; D007251:Influenza, Human / Q000517:prevention & control; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D004220:Disulfides; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines