Cell Culture-derived Influenza Vaccine Research Articles

Efficacy of cell culture-derived influenza vaccines for children: A systematic review and meta-analysis

Efficacy of cell culture-derived influenza vaccines for children: A systematic review and meta-analysis

Resolving Isomeric Structures of Native Glycans by Nanoflow Porous Graphitized Carbon Chromatography-Mass Spectrometry.

Characterization of the structural diversity of glycans by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains an analytical challenge in large-scale glycomics applications because of the presence of heterogeneous composition, ubiquitous isomers, lability of post-translational glycan modifications, and complexity of data interpretation. High-resolution separation of glycan isomers differentiating from positional, linkage, branching, and anomeric structures is often a prerequisite to ensure the comprehensive glycan identification. Here, we developed a straightforward method using self-packed capillary porous graphitic carbon (PGC) columns for nanoflow LC-MS/MS analyses of native glycans released from glycoproteins. The technique enables highly resolved chromatographic separation of over 20 high-mannose glycan isomers in ribonuclease B and a diverse range of hybrid and complex-type sialoglycoforms of fetuin. The distinct structures of anomeric glycans and linkage sialoglycan isomers, α2,3 and α2,6, were identified by the characteristic MS/MS fragment ions. A glycan sequencing strategy utilizing diagnostic ions and complementary fragments specific to branching residues was established to simplify the MS/MS data interpretation of closely related isomeric structures. To promote the PGC-LC-MS/MS-based method for glycome-wide applications, we extended analyses to native sulfoglycans from the egg-propagated and cell culture-derived influenza vaccines and demonstrate the high-resolution separation and structural characterization of underivatized neutral and anionic glycoforms including oligomannosidic glycan anomers, sialoglycan linkage isomers, and regioisomers of afucosylated and fucosylated sulfoglycans containing sulfated-6-GlcNAc and sulfated-4-GalNAc residues.

Immunogenicity and Safety of a Cell Culture-Derived Quadrivalent-Inactivated Influenza Vaccine: A Phase III Randomized Controlled Trial in Adults and Elderly Subjects

Immunogenicity and Safety of a Cell Culture-Derived Quadrivalent-Inactivated Influenza Vaccine: A Phase III Randomized Controlled Trial in Adults and Elderly Subjects

Enhanced production of human influenza virus in PBS-12SF cells with a reduced interferon response

Influenza is one of the most important infectious diseases in humans. The best way to prevent severe illness caused by influenza infection is vaccination. Cell culture-derived influenza vaccines are being considered in addition to the widely used egg-based system in order to support the increasing seasonal demand and to be prepared in case of a pandemic. Cell culture based systems offer increased safety, capacity, and flexibility with reduced downstream processing relative to embryonated eggs. We have previously reported a chick embryo cell line, termed PBS-12SF, that supports replication of human and avian influenza A viruses to high titers (>107 PFU/ml) without the need for exogenous proteases or serum proteins. Viral infections in cells are limited by the Interferon (IFN) response typified by production of type I IFNs that bind to the IFNα/β receptor and activate an antiviral state. In this study, we investigated how neutralizing the interferon (IFN) response in PBS-12SF cells, via shRNA-mediated knock-down of IFNAR1 mRNA expression, affects influenza virus production. We were successful in knocking down ∼90% of IFNAR1 protein expression by this method, resulting in a significant decrease in the response to recombinant chIFNα stimulation in PBS-12SF cells as shown by a reduction in expression of interferon-responsive genes when compared to control cells. Additionally; IFNAR1-knock-down cells displayed enhanced viral HA production and released more virus into cell culture supernatants than parental PBS-12SF cells.

Fabrication of cell culture-derived influenza vaccine dissolvable microstructures and evaluation of immunogenicity in guinea pigs

Microstructure patches provide an opportunity for simple, effective, and safe vaccine administration, while achieving the desired immune response. We have evaluated the MicroCor® transdermal system for cell culture-derived trivalent influenza vaccine administration. Influenza monovalent purified bulk vaccines (monobulks) (H1N1, H3N2, B) were concentrated by tangential flow filtration, lyophilized, and formulated with biocompatible excipients to form the microstructure array dissolvable tips. Standard single radial immunodiffusion (SRID) determined that the influenza antigens retained potency through the formulation and microstructure array fabrication processes. Array stability was evaluated for storage in both refrigerated and room temperature conditions. Microstructure mechanical strength was confirmed by application to excised pig skin, resulting in successful skin penetration and tip dissolution within 5min of microstructure insertion. Guinea pigs immunized with influenza vaccine-loaded microstructures had hemagglutinin inhibition (HI) and IgG titers comparable to those obtained by intramuscular injection. After two immunizations, serum HI titers for all immunized groups were greater than 40 (>4-fold higher than the untreated group). These data demonstrate the feasibility for the development of skin delivery technologies that are compatible with cell culture-derived influenza vaccines.

A flow-through chromatography process for influenza A and B virus purification

Vaccination is still the most efficient measure to protect against influenza virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza virus purification. This study focused on the development of an economic flow-through chromatographic process avoiding virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase® treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for purification of two influenza A virus strains and one influenza B virus strain. The process resulted in high virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase® could be successfully removed from the product fraction.Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines.

Continuous purification of influenza virus using simulated moving bed chromatography

Continuous size exclusion chromatography for the separation of cell culture-derived influenza virus from contaminating proteins was established successfully. Therefore, an open loop simulated moving bed (SMB) setup with one column per zone was applied. Several operating conditions were tested and overall trends were found to be in agreement with expectations derived from theory. Furthermore, the separation performance was compared to an optimized conventional batch chromatography. The yield of influenza virus in the product fraction, based on a hemagglutination assay, was 70% (SMB) and 80% (batch), respectively. The amount of contaminating protein per product was 0.61μgkHAU−1 (SMB) compared to 0.29μgkHAU−1 (batch). This corresponds to a reduction of the respective amount in the feed solution by 60% and 80%, respectively. For both processes, the estimated amount of total protein per vaccine dose would meet the level required for manufacturing of human influenza vaccines prepared in cell cultures. Depending on the strategy chosen for sanitization and equilibration of columns the calculated overall productivity for the SMB process was up to 3.8 times higher compared to the batch mode. SMB, therefore, has the potential to replace single column discontinuous chromatography in order to design more efficient purification trains for production of cell culture-derived influenza vaccines.

Vero cell culture-derived pandemic influenza vaccines: preclinical and clinical development

Several subtypes of influenza A viruses with pandemic potential are endemic in bird populations throughout Asia, Africa and the Middle East, and evidence suggests that these viruses are adapting to the mammalian host. As emphasized by the high mortality rate of humans infected with H5N1 viruses, this situation presents a substantial risk to global human health. The Vero cell culture platform has been used to develop whole-virus influenza vaccines that provide broad cross-clade protection against viruses with pandemic potential, at low antigen doses, without the requirement for adjuvantation. The safety and immunogenicity of these vaccines has been demonstrated in studies with more than 10,000 individuals, including healthy adult and elderly subjects, children and various risk groups. These Vero cell-derived vaccines are licensed for prepandemic and pandemic use. The Vero platform is also being explored to develop next-generation live-attenuated and recombinant vaccines.

A prospective observational safety study on MF59® adjuvanted cell culture-derived vaccine, Celtura® during the A/H1N1 (2009) influenza pandemic

BackgroundThe present study was a prospective observational study to evaluate the safety profile of Celtura®, a monovalent, cell culture-derived, inactivated subunit influenza vaccine prepared from A/California/07/2009(H1N1) with the adjuvant MF59®. Subjects were enrolled prospectively during the H1N1 2009 influenza pandemic at medical centres in Colombia, Chile, Switzerland, and Germany during the period December 2009 to June 2010. MethodsSubjects ages 18 and older were followed for the occurrence of adverse events (AEs) for six months after vaccination. Adverse events of special interest (AESIs) were neuritis, convulsion (seizure), anaphylaxis, encephalitis, vasculitis, Guillain-Barre syndrome, demyelinating conditions, Bell's palsy, and laboratory-confirmed vaccination failure. ResultsOverall, 7348 AEs were reported in 2296 of 3989 enrolled subjects (57.6%). Only two AEs were considered related to injection site reactions. No laboratory-confirmed cases of influenza were reported. There were 108 medically confirmed serious adverse events (SAEs) reported among 73 subjects with 6 such SAEs described as possibly or probably related to vaccination. Three fatal cases were reported and assessed as not related to vaccination. Two AESIs classified as convulsion were reported and assessed as not related to vaccination. Both AESIs occurred well outside the pre-specified 7 day risk window representing the likely timeframe of the occurrence of seizure following vaccination. ConclusionsThe results of this study support the overall good safety profile of MF59 adjuvanted cell culture-derived influenza vaccine as administered in adults during the 2009–2010 H1N1 influenza pandemic. No concern is raised regarding the occurrence of AESIs.

Safety assessment and immunogenicity of a cell-culture-derived influenza vaccine in adults and elderly subjects over three successive influenza seasons

Background Adult and elderly subjects previously immunized with cell culture-derived (CCIV; Optaflu®) or egg-derived (TIV; Agrippal®) trivalent influenza vaccines were enrolled in two extension studies (E1 and E2) to evaluate safety and immunogenicity after revaccination with CCIV/TIV alone or in combination with concomitant pneumococcal vaccine (PV).Methods Adults and elderly subjects (n = 2609) were randomized 1:1 in E1 and allocated 3:1 in E2 to receive CCIV/TIV. In E2, a subset of elderly subjects was randomized to receive CCIV/TIV, with or without PV. Adverse reactions were monitored for six months and immunogenicity was assessed by hemagglutination inhibition (HI) assay using CHMP criteria.Results Overall, the safety profile of both vaccines was similar, no serious adverse events related to either vaccine occurred. Mild or moderate pain was the most commonly reported reaction. Reactogenicity was slightly higher in elderly subjects receiving CCIV/TIV concomitantly with PV [46% vs. 37%; p = non-significant (NS)]. Both vaccines met CHMP licensure criteria for adults and elderly subjects. With concomitant CCIV and PV, all three CHMP criteria were met for A/H1N1 and A/H3N2, whereas the B strain only met seroprotection and GMR criteria.Conclusions Safety and immunogenicity of CCIV was not influenced by the type of vaccine received previously or by concomitant PV administration.

Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production

In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin–Darby canine kidney, PER.C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.

A Cell Culture-Derived Influenza Vaccine Provides Consistent Protection Against Infection and Reduces the Duration and Severity of Disease in Infected Individuals

Current knowledge of the consistency of protection induced by seasonal influenza vaccines over the duration of a full influenza season is limited, and little is known about the clinical course of disease in individuals who become infected despite vaccination. Data from a randomized double-blind placebo-controlled clinical trial undertaken in healthy young adults in the 2008-2009 influenza season were used to investigate the weekly cumulative efficacy of a Vero cell culture-derived influenza vaccine. In addition, the duration and severity of disease in vaccine and placebo recipients with cell culture-confirmed influenza infection were compared. Vaccine efficacy against matching strains was consistently high (73%-82%) throughout the study, including the entire period of the influenza season during which influenza activity was above the epidemic threshold. Vaccine efficacy was also consistent (68%-83%) when calculated for all strains, irrespective of antigenic match. Vaccination also ameliorated disease symptoms when infection was not prevented. Bivariate analysis of duration and severity showed a significant amelioration of myalgia (P = .003), headache (P = .025), and fatigue (P = .013) in infected vaccinated subjects compared with placebo. Cough (P = .143) and oropharyngeal pain (P = .083) were also reduced in infected vaccinated subjects. A Vero cell culture-derived influenza vaccine provides consistently high levels of protection against cell culture-confirmed infection by seasonal influenza virus and significantly reduces the duration and severity of disease in those individuals in which infection is not prevented. ClinicalTrials.gov NCT00566345.

A Cell Culture–Derived Influenza Vaccine Provides Consistent Protection Against Infection and Reduces the Duration and Severity of Disease in Infected Individuals

A Cell Culture–Derived Influenza Vaccine Provides Consistent Protection Against Infection and Reduces the Duration and Severity of Disease in Infected Individuals

Efficacy and correlates of protection for cell culture-derived and egg-derived inactivated influenza vaccines in younger adults

Evaluation of: Barrett PN, Berezuk G, Fritsch S et al. Efficacy, safety, and immunogenicity of a Vero-cell-culture-derived trivalent influenza vaccine: a multicentre, double-blind, randomised, placebo-controlled trial. Lancet 377(9767), 751–759 (2011).The randomized, double-blind, placebo-controlled efficacy study by Barrett et al. demonstrated the efficacy of cell culture-derived trivalent inactivated influenza vaccine (TIV), as manufactured by Baxter BioScience, in healthy adults 18–49 years of age. Efficacy and safety was comparable to that demonstrated by other recent randomized, placebo-controlled studies of cell culture-derived and egg-derived TIV in younger adults. The principal advantage of cell culture manufacturing of influenza vaccine relates to pandemic preparedness, although some advantages exist for seasonal vaccine production. The Barrett study confirmed a correlation between anti-hemagglutinin serum antibody and protection from influenza. However, further analysis of the correlation appears warranted in the Barrett study and in future studies to better describe antibody-induced and vaccine-induced protection from influenza illness.

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

Cell-Culture–Derived Influenza Vaccine Is Safe and Efficacious

Conventional egg-based influenza vaccines have several drawbacks, including a lengthy production period that limits the capacity to match the vaccines

Clinical Efficacy of Cell Culture–Derived and Egg‐Derived Inactivated Subunit Influenza Vaccines in Healthy Adults

More efficient methods are needed to manufacture influenza vaccines. This trial compared the efficacy of cell culture-derived influenza vaccine (CCIV) and egg-derived trivalent inactivated vaccine (TIV) with placebo against laboratory-confirmed influenza illness in healthy adults in the United States, Finland, and Poland during the 2007-2008 influenza season. A total of 11,404 study participants aged 18-49 years were randomized equally to receive CCIV (Optaflu; n = 3828), TIV (Agrippal; n = 3676), or placebo (n = 3900). Each participant was observed during a 6-month study surveillance period. Nasal and throat swabs for virus isolation and characterization were collected from all patients with influenza-like illness. Vaccine immunogenicity was evaluated in a subset of 1045 participants. Efficacy of CCIV and TIV against vaccine-like (83.8% [1-sided 97.5% confidence interval [CI] lower limit, 61.0%] and 78.4% [1-sided 97.5% CI lower limit, 52.1%], respectively) and all circulating influenza virus strains (69.5% [1-sided 97.5% CI lower limit, 55.0%] and 63.0% [1-sided 97.5% lower limit, 46.7%], respectively) exceeded the Center for Biologics Evaluation and Research efficacy criteria. Immunogenicity of both vaccines exceeded the Center for Biologics Evaluation and Research licensing criteria. Both vaccines were well tolerated, with similar safety profiles. Most solicited reactions were mild to moderate in severity and transient. No vaccination-related serious adverse events were reported; no withdrawals resulted from vaccine-related adverse events. Both CCIV and TIV were effective in preventing influenza caused by vaccine-like and by all circulating influenza virus strains, were well tolerated, and had good safety profiles. Both vaccines can be considered for annual influenza vaccination campaigns. NCT00630331.

DNA Depletion by Precipitation in the Purification of Cell Culture‐Derived Influenza Vaccines

AbstractA pilot study for the purification of cell culture‐derived human influenza virus is presented, which focuses on the early removal of DNA by precipitation. Strains of influenza virus were propagated using Madin Darby canine kidney cells as a host. A harvesting time of about 72 h postinfection was chosen to minimize the level of impurities (host cell DNA and protein). Cell culture supernatant was clarified by centrifugation and the performance of this operation was characterized on the basis of Sigma theory. An average clarification efficiency of 93 % (based on turbidity) and a product yield of 85 % (based on hemagglutination activity) were obtained at a load of 1.6 · 10–8 m s–1. Furthermore, the applicability of Sigma theory for scale‐up studies using two different laboratory centrifuges was verified. Selective precipitation of DNA was achieved by the addition of polyethyleneimine (PEI). Full factorial design was applied to optimize selectivity considering pH, ionic strength, and the concentration and molecular weight of PEI. Under optimized conditions, treatment with PEI resulted in a reduction of DNA to 15 % of the initial amount, while 86 % of virions (based on neuraminidase activity) were recovered. The subsequent concentration of virions was realized by tangential‐flow ultrafiltration. Recovery based on hemagglutination activity was determined to 63 % on average. Including the previous precipitation step, overall reduction in DNA after tangential‐flow ultrafiltration was 500‐fold. These results indicate that the suggested unit operations are suited for the early depletion of DNA in cell culture‐derived influenza vaccine production.

Metabolic effects of influenza virus infection in cultured animal cells: Intra- and extracellular metabolite profiling

BackgroundMany details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations.ResultsFor most metabolites the comparison of infected (human influenza A/PR/8/34) and mock-infected cells showed a very similar behavior during the first 10-12 h post infection (pi). Significant changes were observed after about 12 h pi: (1) uptake of extracellular glucose and lactate release into the cell culture supernatant were clearly increased in infected cells compared to mock-infected cells. At the same time (12 h pi) intracellular metabolite concentrations of the upper part of glycolysis were significantly increased. On the contrary, nucleoside triphosphate concentrations of infected cells dropped clearly after 12 h pi. This behaviour was observed for two different human influenza A/PR/8/34 strains at slightly different time points.ConclusionsComparing these results with literature values for the time course of infection with same influenza strains, underline the hypothesis that influenza infection only represents a minor additional burden for host cell metabolism. The metabolic changes observed after12 h pi are most probably caused by the onset of apoptosis in infected cells. The comparison of experimental data from two variants of the A/PR/8/34 virus strain (RKI versus NIBSC) with different productivities and infection dynamics showed comparable metabolic patterns but a clearly different timely behavior. Thus, infection dynamics are obviously reflected in host cell metabolism.

Safety and Immunogenicity of a Novel Influenza Subunit Vaccine Produced in Mammalian Cell Culture

Immunization remains the best prevention strategy for influenza, but production constraints for egg-based influenza vaccines have prompted the development of innovative cell culture manufacturing processes. Here, we describe a novel cell culture-derived influenza vaccine (CCIV) produced in Madin-Darby canine kidney cells. This phase 3, observer-blind, randomized, multicenter study in Poland compared the immunogenicity of a CCIV and a conventional egg-based vaccine. Participants, stratified by age (adults 18-60 years, n = 1300; elderly persons > or = 61 years, n = 1354), received a single intramuscular vaccination. Immunogenicity was assessed 21 days later by hemagglutination inhibition assay. Reactogenicity was assessed using self-completed diary cards. The immunogenicity of CCIV was noninferior to that of the conventional vaccine for all 3 vaccine strains in both age groups, regardless of underlying health status. Both vaccines fulfilled European Union registration criteria and were well tolerated, with similar incidences of solicited local and systemic reactions in both age groups; the only significant difference was an increased frequency of mild or moderate pain with CCIV than the conventional vaccine among adult (22% vs 17%; P < .05) and elderly (9% vs 5%; P < .001) vaccinees. CCIV was well tolerated and highly immunogenic in adults 18 years of age or older. Cell culture may offer greater flexibility of supply during periods of high demand for both seasonal and pandemic vaccines.