Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability
© Feshchenko et al.; licensee BioMed Central Ltd. 2012
Received: 6 July 2012
Accepted: 8 October 2012
Published: 30 October 2012
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.
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.
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.
A novel influenza A virus (H1N1) of swine origin emerged in Mexico and the United States in March and early April 2009. The virus quickly spread worldwide through human-to-human transmission resulting in the World Health Organization raising the influenza pandemic alert to the highest level (phase 6) on June 11, 2009 [1–3]. The outbreak and spread of the first influenza pandemic of the 21st century challenged licensed vaccine manufacturers to rapidly mobilize and generate a prophylactic vaccine. Delivery of initial doses of vaccine to the U.S. public coincided with the second peak of the pandemic, too late to provide timely protection and highlighting the need for alternative production platforms [2, 4].
Two types of licensed influenza vaccines are available in the U.S.: trivalent inactivated vaccine and live attenuated influenza vaccine [5, 6], both produced in embryonated chicken eggs. The process of preparing a new vaccine seed strain suitable for growth in eggs can be quite lengthy, as it involves re-assortment between the genes of a high yielding donor virus, such as A/Puerto Rico/8/34, and the hemagglutinin (HA) and neuraminidase (NA) genes of the new influenza strain . The candidate seed virus strains are then further selected for high growth capability in eggs before they can be used for the production of vaccines. This manufacturing process is not only lengthy but also limited in scalability due to its dependence on the availability of embryonated chicken eggs.
The production of purified recombinant hemagglutinin (rHA) subunit vaccines via the baculovirus expression vector system (BEVS) is a leading alternative platform for influenza vaccine manufacture. The most advanced influenza vaccine candidate produced using this technology (under the trade name Flublok®) is a trivalent composition of three rHA proteins corresponding to the full length HA proteins of the seasonally circulating influenza strains [8–11]. Clinical trials of Flublok have demonstrated that the vaccine is well-tolerated, immunogenic (as assessed by the induction of hemagglutination inhibiting [HAI] antibodies), and provides protection against drifted influenza viruses [8–10]. The rHA proteins in Flublok are produced using genetically modified baculoviruses in lepidopteran insect cells. The proteins are extracted and purified from cell pellet using a combination of filtration and column chromatography methods. Bulk vaccine can be produced within seven weeks of receipt of the HA gene sequence , making it an attractive platform for pandemic vaccine manufacturing as demonstrated during the initial outbreaks of H5N1 .
The original influenza seed viruses used for the egg-based production of the A(H1N1)pdm09 vaccine grew slowly, produced relatively low quantities of HA antigen and showed poor stability [7, 13]. The rHA derived from A/California/07/2009 also revealed differences compared to other rHA proteins, as the pandemic rHA protein was more sensitive to proteolytic degradation and reacted uniquely in the single radial immunodiffusion (SRID) potency assay.
The objectives of this study were (1) to study the properties of the A/California/07/2009 rHA protein and compare it to other rHA proteins derived from seasonal influenza strains and (2) to develop an understanding of the cause of the instability observed with this antigen.
Biochemical and biophysical characterization
Edman sequencing results for H1 A/California/07/2009 trypsin digested fragments
Conserved Cleavage Site
Additional Cleavage Sites
Conserved Cleavage Site
x - unmodified Cys based on the known sequence of H1 A/California/07/2009 rHA
Hemagglutination activity of purified H1 A/California/07/2009, H1 A/Brisbane/59/2007, H1 A/New Caledonia/20/99 and H1 A/Solomon Islands/03/2006 rHA
rHA derived from various H1N1 influenza viruses
Guinea pig red blood cells
Chicken red blood cells
Turkey red blood cells
(HA units/μg protein)
N = 8
N = 6
N = 2
N = 2
Potency by SRID for licensed monovalent H1N1 vaccines calculated using different antisera and reference antigens
SRID reference antigen
Reference antigen source
Potency according to SRID (μg/mL)
SRID antiserum source
Commercial Vaccine 1
Commercial Vaccine 2
Immunogenicity of trivalent vaccine formulations
Vaccine test article
Dose (by SRID)
2008-2009 rHA Formulation
2010-2011 rHA Formulation
2010-2011 Commercial Split Virion Vaccine (FluLaval® Lot # AFLLA599BA)
There was a clear dose dependence of the HAI response to all vaccine components across the test formulations, and the magnitude of the response increased from Day 21 to Day 42 after the second immunization. The immune response generated against A/California/07/2009 (H1) rHA antigen was equal to or slightly greater than that of A/Brisbane/59/2007 (H1) rHA antigen and the commercial egg-based A/California/07/2009 (H1) control by Day 42. The immune responses were also consistent for the H3 and B vaccine antigen components. These results demonstrate that rHA antigens, including A/California/07/2009 rHA, produce a robust immune response.
The A(H1N1)pdm09 influenza vaccine was available late and in short supply as a result of delays in production caused by low yields, poor antigen stability and absence of virus stockpile. Recombinant hemagglutinin-based vaccines are inherently less susceptible to production challenges and are a leading alternative for influenza vaccine manufacture. The most advanced recombinant influenza vaccine candidate is a trivalent formulation of seasonal rHA proteins that can be produced significantly faster than traditional egg-based influenza vaccines and has been shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical trials. Pandemic rHA vaccines hold similar promise.
This in-depth characterization of A/California/07/2009 rHA showed that it is biochemically, biophysically, and antigenically comparable to seasonal rHA antigens. A/California/07/2009 rHA had an electrophoretic mobility similar to that of seasonal rHA proteins and formed higher order, multimeric rosette-like particles of approximately 20–40 nm. This is in contrast to the findings of Khurana et al. who expressed full length A/California/07/2009 HA in a bacterial expression system and found that the purified protein migrated predominantly as a monomer . The reason for this difference is unknown but could relate to the different expression platforms. A/California/07/2009 rHA demonstrated significant biological activity and elicited a strong immune response in mice consistent with that generated by commercial egg-derived A/California/07/2009 vaccine both in this study and previously [19, 20]. Together, these data support the suitability of A/California/07/2009 rHA as a pandemic influenza vaccine alternative. An initial clinical study has confirmed the safety and immunogenicity of this rHA .
The antigenic stability of the A(H1N1)pdm09 vaccine was found to be initially poor . Trypsin digestion of purified A/California/07/2009 rHA uncovered a unique susceptibility of the protein to proteolytic cleavage not found in the seasonal rHA comparators. N-terminal (Edman) sequencing revealed that this cleavage occurs in a subdomain of the HA2 region of the protein that for most HA proteins are predicted to be structured as a coiled-coil. We postulate that the six amino acid changes in this domain in A/California/07/2009 HA may disrupt this structure, leading to decreased antigenic stability. Further studies are needed to determine whether the virus re-assortants ultimately used for egg-based A(H1N1)pdm09 vaccine manufacture possessed modifications that impacted protein structure in this region, improving antigen stability. Preliminary assessment suggests that a purified recombinant rHA derived from re-assortant virus NIBRG-121xp  in fact remained unstable although its interaction with sialic acid receptors was improved (data not shown).
Finally, the antigenic potency of A/California/07/2009 rHA, as determined by the SRID assay, showed dramatic heterogeneity (≥ 2-fold) depending on the assay reagents used. A similar effect was observed for licensed egg-derived monovalent vaccines. This variation indicates that preparation of reagents for potency testing (antisera and reference antigens) with novel pandemic influenza viruses requires further assessment to accommodate recombinant manufacturing strategies available for rapid pandemic response. Moreover, the development of alternative potency assays that are less dependent upon specific antigen-antibody interactions that could be affected by the manufacturing platform is warranted.
These results show that the production of purified recombinant hemagglutinin (rHA) subunit vaccines via the baculovirus expression system is a leading alternative platform for influenza vaccine manufacture. The biochemical, biophysical and immunological characterization of a purified recombinant A/California/07/2009 (H1N1) hemagglutinin has been compared to different seasonal rHA proteins and egg-produced A/California reagents. The data show that purified A/California/07/2009 rHA molecules exist in high molecular weight complexes and form rosette-like particles of 20 – 40 nm in size. Biochemically, the protein exhibits hemagglutination activity and a greater sensitivity to tryptic digestion with additional cleavage in the HA2 subunit. The unique structure of this particular HA antigen may account for poor stability. The A/California/07/2009 rHA protein was found to be antigenically similar to egg-derived virus and showed immunogenicity and development of neutralizing antibody titers in mice.
rHA cloning and baculovirus generation
The H1 A/California/07/2009 cDNA was generated by using influenza viral RNA as a template in a reverse transcriptase PCR reaction (RT-PCR). The cDNA was cloned into baculovirus transfer vector pPSC12, and positive clones were confirmed by DNA sequencing to be identical to the H1 A/California/07/2009 reference sequence (GenBank accession # ACP41953). Spodoptera frugiperda Sf9 cells were co-transfected with linearized Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) genomic DNA and the pPSC12 transfer vector containing H1 A/California/07/2009 cDNA by calcium phosphate precipitation. This method generated recombinant baculoviruses harboring the gene encoding H1 A/California/07/2009 by homologous recombination. Recombinant plaques were isolated and used to generate baculovirus stocks in expresSF+ ® (SF+) insect cells. Further details on the cloning and expression of other rHAs using this system are described elsewhere [9, 22, 23].
rHA protein production
The recombinant baculovirus stock was used to produce the H1 A/California/07/2009 rHA protein. Virus inoculum from the working virus stock was added to 450 L of SF+ insect cell culture in a 600 L bioreactor at a concentration of 2% (v/v) after the insect cells reached a density of 2.0 – 2.5 × 106 cells per mL. The infected culture was incubated at 28°C for 40 – 55 hours and harvested at a viability of 70 - 80%. A cell pellet was generated by centrifugation and the recombinant protein was solubilized using a buffer containing non-ionic detergent. Cells were removed by depth filtration, and the clarified extract was applied to an ion-exchange column. Recombinant HA was eluted and subsequently bound to a hydrophobic interaction column. Following elution, the protein was applied to a Q-membrane to remove any residual DNA. Finally, Q filtrate was diafiltered and the rHA protein formulated in final buffer.
SDS-PAGE and western blot
Proteins were separated using 4-12% NuPAGE Bis-Tris Gels (Cat# NP0323, Life Technologies Corporation, Carlsbad, CA) and 1x MES Running Buffer (50 mM MES, 50 mM Tris, 0.1% sodium dodecyl sulfate, 1 mM EDTA pH 7.3). Non-reducing SDS-PAGE gel samples were prepared using 2x non-reducing disruption buffer (120 mM Tris pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue). Reducing SDS-PAGE gel samples were prepared using 2x disruption buffer (120 mM Tris pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 200 mM dithiothreitol). The SDS-PAGE gels were fixed in pH 1.1 fixative (25% methanol, 10% glacial acetic acid, 10% thrichloroacetic acid) for 10 minutes, followed by staining in Coomassie Blue staining solution (0.1% Brilliant Blue R, 7.7 M reagent alcohol, 1.75 M glacial acetic acid) for 60 minutes. The gels were de-stained in 10% acetic acid. For the Western Blot, the separated proteins were transferred to nitrocellulose membrane (Cat# IB3010, Life Technologies Corporation) or to polyvinylidenefluoride (PVDF) membrane (Cat# IB4010, Life Technologies Corporation) and incubated with a 1:1,000 dilution of A/California/07/2009 (H1N1) influenza antiserum (Lot #09/152, sheep 506/507, NIBSC, UK) followed by a 1:3,000 dilution of anti-sheep IgG (whole molecule) alkaline phosphate secondary antibody (Cat# A5187, Sigma, St. Louis, MO). Recombinant H1 A/California/07/2009 proteins were visualized using chromogenic alkaline phosphatase substrates, 1x NTB (nitro-blue tetrazolium chloride, Cat# 0329, Amresco, Solon, OH) and 1x BCIP (5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt, Cat# 0885, Amresco) in chromagen buffer (2-amino-2-methyl-1-propanol, Cat# 221, Sigma) per the manufacturers’ instructions.
Reaction mixtures containing an rHA concentration of 250 μg/mL were incubated in the absence or presence of 50 μg/mL trypsin (Cat# T1426, Sigma) for 30 min at 2-8°C. Digestion was stopped by heating the samples in 2x disruption buffer. Samples were analyzed by SDS-PAGE and Western blotting, as described above.
The hemagglutination assay was performed in U-bottom 96 well microtiter plates. Recombinant HA antigen (starting concentration of 1 μg/mL; protein concentration determined by BCA [Bicinchoninic Acid; Cat# 23225, Thermo Scientific, Rockford, IL]) was diluted by two-fold serial dilution to a final dilution of 4,096-fold. Fresh red blood cells (RBCs) were washed with 1x PBS (pH 7.2) and then added to the wells. RBCs used include 0.5% chicken RBCs from Charles River SPAFAS, Charleston, SC (Lot #A101213), 1% guinea pig RBCs from ViroMed Laboratories, Minnetonka, MN (Lot#11916), and 0.5% turkey RBCs from ViroMed Laboratories (lot#10984). After one hour of incubation at room temperature, the plates were scored for agglutination. The HA activity is defined by the dilution at which partial agglutination was observed (i.e., 50% of the RBCs were agglutinated or the pellet appeared loose). If only fully agglutinated and/or tight pellets were observed, the endpoint was defined as the average of the dilutions with agglutinated and tight pellets. If no agglutination was observed in any well, then the activity of the test article was deemed less than 20 units/μg of protein.
Size exclusion chromatography (HPLC-SEC)
Size exclusion chromatography (HPLC-SEC) was performed by using an Agilent HPLC System (Agilent Technologies, Santa Clara, CA) with UV or diode array detectors and a Biosuite 450, 8 μm HR SEC column (7.8 × 300 mm) (Waters; Cat#186002166). Samples were run in a mobile phase containing 1x PBS with 300 mM NaCl pH 7.2 at a flow rate of 0.25 mL/min. For each monovalent bulk batch of A/California/07/2009 rHA, 17.5 μg of protein was used. Sizes of rHA multimers were estimated based on a standard curve generated using reagents from a gel filtration HMW calibration kit (Cat# 28-4038-42, GE Healthcare Piscataway, NJ).
Dynamic light scattering
Dynamic Light Scattering was performed on purified rHA protein using a Malvern Zetasizer Nano-S (Malvern Instruments, Worcestershire, UK) according to the manufacturer’s instructions. Zetasizer software (Version 6.20) was used for data analysis. Bulk drug substance was analyzed without dilution by adding 60–70 μL of sample to a microcuvette and reading (typically) three sets of 12–14 individual scans. The volume-average size distributions were averaged and binned to <20 nm, 20–40 nm, and >40 nm.
Transmission Electron Microscopy was performed by Nanoimaging Services (La Jolla, CA). Samples were prepared using a continuous carbon grid method with grids of nitrocellulose supported 400-mesh copper. Three microliters of 1:100 diluted samples (~6 μg/mL protein) were applied to a cleaned grid, blotted with filter paper, and immediately stained with uranyl formate. Electron microscopy was performed using an FEI Tecnai T12 electron microscope, operating at 120KeV, equipped with an FEI Eagle 4 K × 4 K CCD camera using nominal magnifications of 110,000x (0.10 nm/pixel), 52,000x (0.21 nm/pixel), and 21,000x (0.50 nm/pixel) at electron doses of approximately 10–15 e/Å2. Negative stain grids were transferred into the electron microscope using a room temperature stage.
Approximately 6 μg of trypsin treated or untreated A/California/07/2009 rHA was loaded on an SDS-PAGE gel and run under reducing conditions. Protein bands were transferred to a PVDF membrane and stained with 0.1% Coomassie Blue R-250 in 40% methanol and 1% acetic acid, followed by de-staining with 50% methanol and water. Protein bands of interest were excised from the PVDF membrane and submitted to the Protein Core Facility at Columbia University (New York, NY) for N-terminal (Edman) sequencing. The stained bands were sequenced on an Applied Biosystems 494 protein sequencer according to the manufacturer’s instructions.
Single radial immunodiffusion (SRID) assay
SRID assays were performed as described previously [17, 24]. Briefly, an antibody solution at the optimal working concentration was mixed with melted 1% agarose (Cat# 50010, SeaKem ME, Lonza, Rockland, ME ) in 1x PBS (pH 7.2) (Cat# 20012–050, Life Technologies Corporation) at 54° - 56°C. Following solidification on GelBond film (Cat# 53734, Lonza, Rockland, ME) at room temperature, 4 mm wells were punched in the gels. Initial dilutions of test samples and reference standards were prepared in 1% Zwittergent 3–14 (Cat# 693017, Calbiochem, Darmstadt, Germany), incubated for 30 min at room temperature, and further diluted with 1% Zwittergent 3–14 in PBS. Twenty microliter samples were then applied to the agarose wells. The gels were placed in a sealed moist chamber at room temperature for 18 hours. Following incubation, the gels were washed first with 1x PBS (pH 7.2) and then water, dried and stained with Coomassie Brilliant Blue R250 (Cat# BO149, Sigma). After de-staining, the gels were dried, and SRID ring diameters were measured using the GT Vision SRID Reading Program (GT Vision LLC, Hagerstown, MD). The diameters of the precipitin rings were measured in two orthogonal directions. Recombinant HA potency was calculated in μg/mL by the parallel line bioassay method using reference and test rHA antigen response curves (log antigen dilution vs. log zone diameter). Statistical parameters for determining test validity were based on correlation coefficients (r ≥ 0.95) and the equality of slopes (t < 4.604) between test and reference antigens. NIBSC antiserum lot 09/152, CBER experimental antiserum lot H1-Ab-1004 and Protein Sciences Protein G purified rabbit antiserum were used in the SRID assay. In addition, X-181 CBER antigen lot H1-Ag-1002, X-181 NIBSC antigen lot 09/294, X-179A CBER antigen lot 69, X-179A NIBSC antigen lot 09/174, and two different purified rHA lots were used. Licensed (egg-derived) monovalent A/California/07/2009 H1N1 pandemic vaccines from two manufacturers were also evaluated (Sanofi Pasteur Lot UP088AA and Novartis Lot 110739).
Generation of rabbit polyclonal antibody
New Zealand White Rabbit immunization was performed under contract with Harlan Bioproducts for Science, Inc. (Madison, Wisconsin). An approved 112 day animal protocol was used. Recombinant H1 A/California/07/2009 was used as an antigen. Freund’s adjuvant was used to generate the rabbit antiserum. Approximately 2 mg of antigen at a minimum protein concentration of 0.5 mg/mL was used per rabbit. The rabbit antiserum from the final bleed was purified over a Protein G Sepharose column (Cat# 17-0618-05, GE Healthcare) per the manufacturer’s recommendations. The eluted antibody fraction was dialyzed against 1x PBS (pH 7.2) (Cat# 20012–050, Life Technologies Corporation). The purified rHA H1 A/California/07/2009 antibody was used in the SRID assay. All work was conducted ethically, with animal welfare as a top priority, in full compliance with national animal welfare regulations, under PHS Assurance in a fully USDA licensed facility.
Animal immunization and HA inhibition (HAI) assays
The Animal Core Facility at Colorado State University (CSU) conducted the immunization and determination of HAI titers. Briefly, 6 – 8 week old female CD1 mice were administered trivalent vaccine formulations containing purified recombinant H1 A/California/07/2009 rHA, H1 A/Brisbane/59/2007 rHA, or an A/California/07/2009 egg-based commercial vaccine in trivalent formulation (FluLaval®, GSK, Lot # AFLLA599BA, multi-dose formulation) by intramuscular (IM) injections. The formulated doses were based on SRID.
For HAI titer determination, individual serum samples were treated with receptor destroying enzyme (RDE, from Vibrio cholera Denka-Seiken, Tokyo, Japan) to remove non-specific inhibitors and tested against 4 hemagglutination units (HAU) of the respective influenza viruses grown in eggs using 0.5% chicken RBCs as previously described . All serum samples were tested in duplicate at a 1:10 starting dilution. The HAI titer was defined as the reciprocal of the greatest dilution that completely inhibited the agglutination of the chicken RBCs. A titer value of 5 was assigned to represent responses below the assay detection limit. All work was performed ethically in compliance with all federal, state, and local laws, regulations, and policies, as well as Colorado State University internal policies http://web.research.colostate.edu/ACP/Regulations.aspx.
We thank Peter DiMauro and Michelle Sorensen for help with protein purification and analytics, respectively. This work was supported by contract HHS0100200900106C from the Biomedical Advanced Research and Development Authority (BARDA), Department of Health and Human Services.
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