title: Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss.
abstract: BACKGROUND
  Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form.
  
  
  RESULTS
  The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach.
  
  
  CONCLUSIONS
  rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Holtz
authors.forename: Pamela S
authors.initials: PS
authors.lastname: Robinson
authors.forename: Erin E
authors.initials: EE
authors.lastname: Matthews
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Clifton E
authors.initials: CE
authors.lastname: McPherson
authors.forename: Nikolai
authors.initials: N
authors.lastname: Khramtsov
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Reifler
authors.forename: Jamal
authors.initials: J
authors.lastname: Meghrous
authors.forename: David G
authors.initials: DG
authors.lastname: Rhodes
authors.forename: Manon M
authors.initials: MM
authors.lastname: Cox
authors.forename: Indresh K
authors.initials: IK
authors.lastname: Srivastava
mesh_terms: D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007252:Influenza Vaccines / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D007251:Influenza, Human / Q000276:immunology / Q000517:prevention & control / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins; D003545:Cysteine