[show abstract][hide abstract] 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.
[show abstract][hide abstract] ABSTRACT: Alternative methods for influenza vaccine production are needed to ensure adequate supplies.
Healthy adults 50-64 years were assigned randomly to receive one intramuscular injection of trivalent recombinant hemagglutinin (rHA) or U.S. licensed trivalent inactivated vaccine (TIV) containing H1, H3 and B antigens (Ag) derived from 2007 to 2008 influenza virus strains A/Solomon Islands/03/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Each rHA dose contained 45 μg HA/strain of the 2007-2008 FDA-recommended Ag vs. 15 μg/strain for TIV. Antibody (Ab) responses were measured using a hemagglutination-inhibition (HAI) assay at baseline and 28 days post-vaccination. Respiratory samples for viral culture were collected from subjects with influenza-like illness (ILI) during the 2007-2008 season in the U.S.
601 subjects were enrolled. Vaccines were well tolerated. Seroconversion (the percentage of subjects with either (a) a pre-vaccination HAI titer ≤ 10 and a post-vaccination HAI titer ≥ 40 or (b) a pre-vaccination titer ≥ 10 and a minimum four-fold rise in post-vaccination HAI antibody titer) in the TIV and rHA groups, respectively, was obtained in 66% vs. 72% for H1; 44% vs. 61% for H3; and 41% vs. 41% for B. Proportions achieving titers ≥ 40 were 96% vs. 96% for H1, 75% vs. 85% for H3, and 94% vs. 93% vs. B. Geometric mean titer ratios at day 28 (TIV/rHA) were 0.77 for H1; 0.58 for H3; and 1.05 for B, respectively. ILI frequencies were low and similar in both groups.
Both vaccines were safe and immunogenic. Ab responses vs. H1 and H3 Ags were significantly higher in the rHA group, with similar responses to B. Furthermore, the FluBlok group had a statistically significantly higher seroconversion rate against influenza A/H3N2 compared to the TIV group.
[show abstract][hide abstract] ABSTRACT: The utility of a new instrument for rapid virus quantitation, the Virus Counter, was evaluated in a blind study conducted at three sites. This instrument is a substantially improved version of the original academic research instrument described previously by Stoffel and Rowlen (2005a). The addition of hydrodynamic focusing, a self-contained fluidics system and customized software for system control and data analysis has resulted in a commercially viable and available design. Baculovirus samples were provided by Protein Sciences Corporation and blinded to InDevR and Baylor College of Medicine. Protein Sciences Corporation and Baylor College of Medicine analyzed the samples by plaque assay and InDevR analyzed the samples using the Virus Counter. Serial dilution of stock viruses into growth media and buffer allowed for comparison of measured versus intended concentrations. Direct log-scale comparison between pooled Virus Counter results and pooled plaque assay results indicated a linear relationship (slope=1.1±0.2, R(2)=0.86) with statistically significant Pearson correlation (r=0.93, p<0.001).
Journal of virological methods 10/2010; 171(1):111-6. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alternative substrates for influenza vaccine production are needed to ensure adequate supplies. We evaluated the relative safety and immunogenicity of recombinant hemagglutinin (rHA) or trivalent inactivated vaccine (TIV) among 869 > or =65-year-old subjects in a randomized clinical trial. Virologic surveillance for influenza-like illness (ILI) was conducted during the 2006-2007 epidemic. Vaccines were well tolerated. Seroconversion rates vs. influenza A/H1N1 and H3N2 antigens were superior in the rHA group, but were inferior vs. influenza B; however, results for influenza B are confounded since the vaccine antigens were different. ILI frequencies were low and similar in both groups. Studies assessing relative immunogenicity of vaccines using identical B Ags are warranted.
[show abstract][hide abstract] ABSTRACT: A robust and reliable GMP-compatible fed-batch process was successfully developed for the production of recombinant hemagglutinin (rHA) proteins by expresSF cells. The feeding solution, feeding strategy as well as the cell density at infection were optimized to maximize the final rHA production yields without affecting the existing rHA recovery protocol and downstream process. A simple and stable feeding solution was formulated and a rational feeding regimen designed to yield, depending on the rHA baculovirus used, between 2- and 3-fold enhancements in volumetric rHA production with increased specific productivity compared to the batch culture. Recombinant HA from fed-batch cultures could be simply recovered following cell lysis and purified through chromatographic steps. Overall, the increased rHA yield was maintained throughout the whole process. The performance, reproducibility and scalability of the fed-batch process was successfully demonstrated in 12 bioreactor runs of 2- and 10-L working volume using five different rHA encoding baculoviruses.
[show abstract][hide abstract] ABSTRACT: FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects. The insect cell-baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus-insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok. In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.
[show abstract][hide abstract] ABSTRACT: FluBlok, a recombinant trivalent hemagglutinin (HA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV) manufacturing process. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. This review discusses the four main clinical studies that were used to support licensure of FluBlok under the 'Accelerated Approval' mechanism in the United States.
Influenza and Other Respiratory Viruses 12/2008; 2(6):211-9. · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: The baculovirus insect cell production technology allows for rapid vaccine production and is particularly suitable for influenza vaccines where annual adjustment of the composition is required. Recombinant hemagglutinin produced using this technology is safe, immunogenic and effective in preventing cell-culture confirmed influenza in individuals; recombinant neuraminidase may play a role as an additive to improve the currently licensed influenza vaccines. Universal vaccine candidates, such as matrix protein M2 and nucleocapsid protein, are yet to enter the clinic whereas the first pandemic influenza virus-like particle (VLP) vaccine candidate is in clinical development. This review presents an overview of the use of this production system for the development of various influenza vaccine targets, including hemagglutinin, neuraminidase, M2, nucleoprotein and VLPs containing multiple influenza proteins. The development progress and the advantages and disadvantages of each vaccine candidate are discussed.
Current opinion in molecular therapeutics 03/2008; 10(1):56-61. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: A recombinant trivalent hemagglutinin (HA) vaccine produced in cell culture using the baculovirus expression system provides an attractive alternative to the current egg-based influenza vaccine (Trivalent Inactivated Influenza Vaccine [TIV]) manufacturing process. The HA genes from the annual World Health Organization-recommended strains are cloned, expressed, and purified using a general purification process. Here, we provide an overview of the expression technology used to make the annual adjustment of the vaccine and the clinical studies completed to date with recombinant HA. The highly purified protein vaccine, administered at three times higher antigen content than TIV, results in stronger immunogenicity, a long-lasting immune response and provides cross-protection against drift variant influenza viruses. Furthermore, the vaccine does not contain egg proteins, adjuvants, preservatives, endotoxins, or antibiotics and can therefore be used in a broader population.
Influenza and Other Respiratory Viruses 02/2007; 1(1):35-40. · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Influenza-virus hemagglutinin (HA) protein expressed in insect cells by recombinant baculovirus is a candidate influenza vaccine.
In a randomized, double-blind trial conducted in 399 adults > or = 65 years of age, the efficacy of trivalent inactivated influenza vaccine (TIV) licensed for intramuscular injection was compared with that of trivalent baculovirus-expressed HA vaccine administered at doses of 15 microg, 45 microg, or 135 microg of each HA.
Compared with TIV, baculovirus-expressed HA vaccine was safe and induced better serum antibody responses to the H3 component when administered at doses of 45 microg or 135 microg of each HA.
Baculovirus-expressed HA is a safe and immunogenic influenza vaccine in elderly adults.
The Journal of Infectious Diseases 06/2006; 193(9):1223-8. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: A recombinant SARS-CoV spike (S) glycoprotein vaccine produced in insect cells in a pre-clinical development stage is described. A truncated version of S glycoprotein, containing only the ecto-domain, as well as a His-tagged full-length version were cloned and expressed in a serum-free insect cell line, ExpresSF+. The proteins, purified to apparent homogeneity by liquid column chromatography, were formulated without adjuvant at 3, 9, 27, and 50 microg per dose in phosphate saline and used to immunize mice. Both antigens in each formulation elicited a strong immune response after two or three vaccinations with the antigen. Neutralizing antibody titers correlated closely with standard ELISA reactivity against the S glycoprotein. The truncated S protein was also formulated with an adjuvant, aluminum hydroxide, at 1 microg per dose (+/-adjuvant), and 5 microg per dose (+/-adjuvant). Significantly enhanced immune responses, manifested by higher titers of serum ELISA and viral neutralizing antibodies, were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein. These findings indicate that the ecto-domain of SARS-CoV S glycoprotein vaccine, with or without adjuvant, is immunogenic and induces high titers of virus neutralizing antibodies to levels similar to those achieved with the full S glycoprotein vaccine.
[show abstract][hide abstract] ABSTRACT: Numerous human infections with avian influenza viruses in Asia in recent years have raised the concern that the next influenza pandemic is imminent. The most effective way to combat influenza is through the vaccination of the public. However, a minimum of 3-6 months is needed to develop an influenza vaccine using the traditional egg-based vaccine approach. The influenza hemagglutinin protein (HA), the active ingredient in the current vaccine, can be expressed in insect cells using the baculovirus expression vector system and purified rapidly. An influenza vaccine based on such a recombinant antigen allows a more timely response to a potential influenza pandemic. Here, we report an innovative monitoring assay for recombinant HA (rHA) expression and a rapid purification process. Various biochemical analyses indicate that the purified rHA is properly folded and biologically active.
[show abstract][hide abstract] ABSTRACT: Pandemic influenza has become a high priority item for all public health authorities. An influenza pandemic is believed to be imminent, and scientists agree that it will be a matter of when, where, and what will be the causative agent. Recently, most attention has been directed to human cases of avian influenza caused by a H5N1 avian influenza virus. An effective vaccine will be needed to substantially reduce the impact of an influenza pandemic. Current influenza vaccine manufacturing technology is not adequate to support vaccine production in the event of an avian influenza outbreak, and it has now become clear that new innovative production technology is required. Antiviral drugs, on the other hand, can play a very important role in slowing the disease spread but are in short supply and resistance has been a major issue. Here, we provide an update on the status of pandemic vaccine development and antiviral drugs. Finally, we conclude with some proposed areas of focus in pandemic vaccine preparedness.
The Yale journal of biology and medicine 11/2005; 78(5):321-8.
[show abstract][hide abstract] ABSTRACT: An overview of influenza vaccines in development is provided, with an emphasis on new cell-based protein vaccine candidates. The current licensed vaccine is a cost-effective means to reduce the impact of influenza with a known mechanism of action. Most vaccine companies are focusing on the production of whole influenza viruses in various cell lines to replace egg-based manufacturing technology. Only a limited number of targets have been identified for the development of cell-based protein influenza vaccines. They include hemagglutinin, neuraminidase, M2 and nucleocapsid protein. These protein-based vaccine candidates are discussed, along with their progress in clinical development and the advantages and disadvantages of each vaccine approach.
Current opinion in molecular therapeutics 03/2005; 7(1):24-9. · 3.42 Impact Factor