Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

Abstract

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently by using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially-expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

Oligomeric Recombinant H5 HA1 Vaccine Produced in Bacteria Protects Ferrets from Homologous and Heterologous Wild-Type H5N1 Influenza Challenge and Controls Viral Loads Better than Subunit H5N1 Vaccine by Eliciting High-Affinity Antibodies

“…We showed previously that it is possible to generate a functional oligomeric rHA1 domain in the absence of the HA2 stalk domain (16,20,21,22). We further analyzed the impact of the oligomeric versus monomeric H5N1 HA1 proteins on immunogenicity and protective efficacy of the globular domain and evaluated the quality of antibodies generated in ferrets.…”

“…Electron microscopy analysis was essentially performed as previously described (16). Briefly, after negative staining of the sample for 2 min with 1% uranyl acetate, samplebearing grids were analyzed in a CM120 transmission electron microscope (FEI, Hillsboro, OR) operating at 120 kV.…”

“…However, it was not known if nonglycosylated proteins would present antigenically relevant epitopes. Recently, we demonstrated that bacterially produced influenza HA1 domains (amino acids [aa] 1 to 320) from several pandemic strains are properly folded, form functional oligomers that can agglutinate red blood cells (RBC), and elicit broadly neutralizing antibodies upon immunization (16,20,21). The oligomerization signal was mapped to the first five amino acids in the N terminus of HA1 (20).…”

H5N1 Virus-Like Particle Vaccine Elicits Cross-Reactive Neutralizing Antibodies That Preferentially Bind to the Oligomeric Form of Influenza Virus Hemagglutinin in Humans

“…In previous studies, we demonstrated that the HA1 globular domains of multiple influenza virus strains can form functional trimers and oligomers in the absence of HA2 (17,20,21). The oligomerization was dependent on the first 8 amino acids in the N terminus of HA1 (20).…”

Collaborative studies on the development of national reference standards for potency determination of H7N9 influenza vaccine

“…The similar studies have been previously reported by scientists from CBER, FDA. 16,17 There have been many reports in recent years on the development of alternative methods for the potency analyses of the vaccine. Garcia et al reported the applicability of 2D-HPLC for the characterization of vaccine proteins.…”