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 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.

FIGURE 1. Characterization of purified H1N1pdm09 rHA1 protein from E. coli and H1N1pdm09 vaccine by gel filtration chromatography, transmission electron microscopy, hemagglutination, and SRID assay following rHA1 adsorption

Superdex S-200 gel filtration chromatography of bacterial H1N1pdm09 rHA1 and H1N1pdm09 vaccine. Purified H1N1pdm09 HA1 with intact N-terminus (1–320) (A) and H1N1pdm09 vaccine (B) from the reassorted virus strain were subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). The elution volumes of protein species are shown. (C) Negative stain electron micrograph (top) and class average images (bottom) of bacterially purified H1N1pdm09 HA1 protein oligomeric forms. Oligomers showing a clear rosette-like structure are encircled. Scale bars, 20 nm. (D) Agglutination of human RBC by properly folded bacterial H1N1pdm09 rHA1 (1–320) protein along with H1N1pdm09 vaccine. Serial dilutions of purified rHA1 were mixed with washed RBC and hemagglutination was read after 60 min at RT. Reassorted H1N1pdm09 virus was used as a positive control. H1N1pdm09 vaccine was used at a starting concentration of 15µg/ml. Dilutions of H1N1pdm09 potency reference antigen were analyzed by SRID assay using NIBSC (anti-H1N1pdm09) reference antiserum before (E) and after (F) adsorption with bacterially produced H1N1pdm09 rHA1.

Figure 2. SRID analysis of H1N1pdm09 potency reference antigen using NIBSC (anti-H1N1pdm09) reference antiserum and antiserum prepared by the alternative method using the bacterially purified rHA1-H1N1pdm09

Dilutions of A/California/7/2009 (H1N1pdm09) or A/Brisbane/59/2007 (H1N1) reference antigens were analyzed by SRID using either the homologous NIBSC reference antiserum (A) or the alternative CBER rHA1-sheep antiserum (B). (C) Comparison of NIBSC and CBER anti-rHA1 H1N1pdm09 sheep sera. Precipitin rings in the SRID assay were measured in two directions to the nearest 0.1 mm for determination of diameter with the serial dilutions of H1N1pdm09 reference antigens with the NIBSC sheep sera (in black) and the two sheep [Sh-270 (in red) & Sh-7870 (in blue)] immunized with bacterially purified rHA1. Slopes and correlation coefficient calculated for each sheep sera are represented in their respective colors.

Figure 3. Heat treatment of H1N1pdm09 reference antigen to evaluate antibody specificity by SRID using NIBSC (anti-H1N1pdm09) reference antiserum and rHA1-H1N1pdm09 sheep antiserum

Dilutions of H1N1pdm09 (A/California/7/2009) reference antigen were analyzed by SRID assay before or after heat treatment at 56°C for 3 hours, using either the homologous NIBSC reference antiserum (A) or the alternative CBER rHA1-sheep antiserum (B).

Figure 4. H5N1 rHA1 (1–320) from A/Vietnam/1203/04 and A/Indonesia/05/2005 elicits high titer cross-neutralizing antibodies and generate H5N1 specific SRID antiserum in rabbits

(A) Animals were immunized with 100 µg proteins mixed with TiterMax adjuvant every three weeks. Sera were collected 8 days after each vaccination and analyzed in a microneutralization assay against various H5N1 virus strains. Representative of three experiments. Dilutions of A/Vietnam/1203/04 (H5N1; Clade-1), A/Indonesia/05/2005 (H5N1; Clade-2),, A/Victoria/210/2009 (H3N2) or A/California/7/2009 (H1N1pdm09) reference antigens were analyzed by SRID using either the CBER rHA1-A/Vietnam/1203/04 antiserum (B) or the CBER rHA1-A/Indonesia/05/2005 antiserum (C). Parallel line bioassay method for comparison of traditional anti-H5N1 sheep sera with either CBER rHA1-A/Vietnam/1203/04 antiserum (D) or the CBER rHA1-A/Indonesia/05/2005 antiserum (E). Precipitin rings in the SRID assay were measured in two directions to the nearest 0.1 mm for determination of diameter with the serial dilutions of homologous H5N1-reference antigens with the traditional anti-H5N1 sheep sera (in black) and the rabbits immunized with bacterially purified H5N1-rHA1. Correlation coefficient calculated for each animal serum is represented in their respective colors.