Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability

Abstract

Background: The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinant technology offers the opportunity to shorten manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine candidate for seasonal influenza produced using the baculovirus expression vector system (BEVS) was shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical studies. In this study, we describe the characterization of the A/California/07/2009 rHA protein and compare the H1N1 pandemic rHA to other seasonal rHA proteins.

Results: Our data show that, like other rHA proteins, purified A/California/07/2009 rHA forms multimeric rosette-like particles of 20-40 nm that are biologically active and immunogenic in mice as assayed by hemagglutination inhibition (HAI) antibody titers. However, proteolytic digest analysis revealed that A/California/07/2009 rHA is more susceptible to proteolytic degradation than rHA proteins derived from other seasonal influenza viruses. We identified a specific proteolytic site conserved across multiple hemagglutinin (HA) proteins that is likely more accessible in A/California/07/2009 HA, possibly as a result of differences in its protein structure, and may contribute to lower antigen stability.

Conclusion: We conclude that, similar to the recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine is likely to perform comparably to licensed A(H1N1)pdm09 vaccines and could offer manufacturing advantages.

Figure 1

Reducing and non-reducing SDS-PAGE of H1N1 rHA proteins. For each sample, the respective purified rHA protein was diluted to a concentration of 100 μg/mL in reducing or non-reducing SDS-PAGE sample buffer, and 1 μg was loaded per lane. The samples were separated using 4 – 12% gradient Nu-PAGE gels and stained with Coomassie Blue. HA0 represents full-length rHA protein and HA1 and HA2 peptide fragments of HA0. Molecular weights of proteins are shown in kilodaltons. rHA proteins were produced by Protein Sciences Corporation.

Figure 2

HPLC-SEC chromatograms of rHA proteins. Volumes corresponding to 17.5 μg of the indicated rHA proteins were injected onto a Biosuite 450 size exclusion column as described in Materials and Methods. Retention times for the respective rHA proteins were 36.0 minutes (A/California); 31.4 minutes (B/Brisbane) and 31.1 minutes (A/Perth).

Figure 3

Size determination of rHA complexes by Dynamic Light Scattering. The results show the percentage of rHA protein in the size increments indicated. The results plotted are means of volume-average diameters in the specified size range obtained from multiple lots (A/California/07/2009 N = 5; A/Brisbane/59/2007 N = 3; A/Perth/16/2009 N = 4; B/Brisbane/60/2008 N = 4).

Figure 4

Transmission Electron Microscopy images of purified rHA proteins. All images were obtained at nominal 52,000× magnification. The white scale bar represents 200 nm.

Figure 5

Trypsin digestion of rHA proteins. rHA proteins were digested with 50 μg/mL trypsin for 30 minutes at 2-8°C. Approximately 1 μg of rHA protein was loaded per lane under reducing conditions. The two additional HA2 peptides (HA2a and HA2b) are indicated with arrows. Molecular weights of proteins are shown in kilodaltons. Panel A: Coomassie Blue-stained SDS-PAGE gel. Panel B: Western blot of trypsin-digested H1 A/California/07/2009 rHA protein. rHA protein was visualized using anti-H1N1 A/California/07/2009 serum from NIBSC (lot 09/152; sheep 506/507).

Figure 6

CLUSTAL multiple sequence alignment and secondary structure prediction for H1 A/California/07/2009, H1 A/New Caledonia/20/99, H1 A/Brisbane/59/2007 and H1 A/Solomon Islands/03/2006. A structure prediction was established using ExPASy Proteomics Tools (http://sequerome.georgetown.edu/sequerome, secondary structure prediction). The conserved cleavage site at R-324 is shown with an asterisk. The additional trypsin cleavage region is over-lined in red. The cleavage site K-419 identified from the digested fragments is indicated by a red arrow. Additional cleavage sites R-420, K-426 and K-427 are indicated by black arrows. The predicted coiled-coil region is indicated by the green over-line. The transmembrane domain is shown by the blue over-line. Amino acids that are divergent from the H1 rHA consensus sequence are highlighted in black. H1 A/California/07/2009 amino acid numbering is based on full length sequence.

Figure 7

SRID comparison of recombinant and egg-derived HA proteins and anti-serum against H1 A/California/07/2009. Reference antigens are identified by re-assortant (X-181 or X-179A) and source (CBER or NIBSC), and were diluted to 30 μg/mL based on the concentrations provided in the product circulars. Two licensed H1N1 A/California/07/2009 monovalent vaccines (Novartis [1] and Sanofi Pasteur [2]) were included in this analysis. The A/California/07/2009 rHA lots were diluted to a target of 30 μg/mL based on total protein measurements. Panel A: SRID gel using sheep antiserum obtained from NIBSC generated against egg-derived A/California/07/2009 HA; Panel B: SRID gel using CBER experimental sheep antiserum generated against an E. coli-expressed HA1 domain of A/California/07/2009 HA; Panel C: SRID gel using Protein Sciences Corporation (PSC) experimental rabbit antiserum generated against BEVS-insect cell derived rHA.