Influenza vaccines have been prepared in embryonated chicken eggs and used for more than 60 years. Although this older technology is adequate to produce hundreds of millions of doses per year, most viral vaccines are now being produced in cell culture platforms. The question of whether egg-based influenza vaccines will continue to serve the needs of the growing influenza vaccine market is considered here. In 2006, the US government committed to support the development of cell-based influenza vaccines by funding advanced development and expansion of domestic manufacturing infrastructure. Funding has also been provided for other recombinant DNA approaches that do not depend on growth of influenza viruses. As the influenza vaccine industry expands over the next 5-10 years, it will be interesting to follow which of these various technologies are able to best meet the needs of a growing influenza vaccine market.

The viral surface protein hemagglutinin (HA) has been recognized as a key antigen in the host response to influenza virus in both natural infection and vaccination because neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system can be used for the production of recombinant HA molecules and is suitable for influenza vaccine production where annual adjustment of the vaccine is required. This expression system is generally considered safe with minimal potential for growth of human pathogens. Extensive characterization of this novel cell substrate has been performed, none of which has revealed the presence of adventitious agents. Multiple clinical studies have demonstrated that the vaccine is safe, well-tolerated and immunogenic. The baculovirus-insect cell system could, therefore, be used for the expedited production of a safe and efficacious influenza vaccine. As a result, this technology should provide a fast track worldwide solution for newly emerging influenza strains or pandemic preparedness within a few years.

An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.

Over the last decade, adherent MDCK (Madin Darby canine kidney) and Vero cells have attracted considerable attention for production of cell culture-derived influenza vaccines. While numerous publications deal with the design and the optimization of corresponding upstream processes, one-to-one comparisons of these cell lines under comparable cultivation conditions have largely been neglected. Therefore, a direct comparison of influenza virus production with adherent MDCK and Vero cells in T-flasks, roller bottles, and lab-scale bioreactors was performed in this study. First, virus seeds had to be adapted to Vero cells by multiple passages. Glycan analysis of the hemagglutinin (HA) protein showed that for influenza A/PR/8/34 H1N1, three passages were sufficient to achieve a stable new N-glycan fingerprint, higher yields, and a faster increase to maximum HA titers. Compared to MDCK cells, virus production in serum-free medium with Vero cells was highly sensitive to trypsin concentration. Virus stability at 37 degrees C for different virus strains showed differences depending on medium, virus strain, and cell line. After careful adjustment of corresponding parameters, comparable productivity was obtained with both host cell lines in small-scale cultivation systems. However, using these cultivation conditions in lab-scale bioreactors (stirred tank, wave bioreactor) resulted in lower productivities for Vero cells.

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An Madin-Darby canine kidney (MDCK) cell line, 9B9-1E4, was cloned by limit dilution from a heterologous cell population and chosen as a potential production cell substrate for cell culture-based influenza vaccine manufacture. Since MDCK cells are transformed cells of canine origin, extensive characterization, including evaluation of tumorigenicity and oncogenicity, was performed to ensure the safety of this cell line for vaccine production. Injection of intact MDCK cells into adult and newborn athymic nude mice did not lead to progressive tumor formation in two separate tumorigenicity studies. In addition, neither MDCK cell lysate nor cellular DNA induced tumors in newborn rodents (athymic nude mice, hamsters and rats) in six oncogenicity studies. Observations from these studies demonstrate the low tumorigenic and oncogenic potential of the MDCK cell clone 9B9-1E4. These observations coupled with other characterization study results strongly suggest a high safety assurance level can be achieved through cell cloning and selection of low tumorigenic and oncogenic cells for influenza vaccine production.

BACKGROUND This study assessed the safety, reactogenicity, and immunogenicity of an injectable cell culture-derived influenza vaccine (CCIV), compared with those of an injectable egg-based trivalent inactivated influenza vaccine (TIV). METHODS Adult subjects (n = 613; 18 to <50 years of age) were randomized (1:1) to receive either CCIV or TIV. The safety and reactogenicity of the 2 vaccines were assessed on the basis of solicited indicators and other adverse events (AEs) within 7 days of vaccination. All serious AEs and those AEs resulting in withdrawal were recorded throughout the study. Antibody titers were determined by the hemagglutination inhibition assay, using egg- and cell-derived antigens. Immunogenicity was assessed on the basis of the ratio of postvaccination (day 22) geometric mean titers (GMTs) between the 2 vaccines, seroprotection rates, and seroconversion rates. RESULTS There was no clinically relevant difference between the safety and reactogenicity profiles of the 2 vaccines. The immunogenicity of CCIV was demonstrated to be noninferior to that of TIV on the basis of the ratio of postvaccination GMTs between the 2 vaccines. GMTs, seroprotection rates, and seroconversion rates were comparable between the 2 vaccines. CONCLUSIONS The safety, reactogenicity, and immunogenicity of the CCIV and the egg-based TIV are comparable.

Few suspension cells can be used for vaccine manufacturing today as they either do not meet requirements from health regulatory authorities or do not produce high virus titres. Two new avian designer cell lines (AGE1.CR and AGE1.CR.pIX) that have been adapted to grow in suspension in serum-free medium were evaluated for their potential as host cells for influenza and modified vaccinia Ankara (MVA, wild type) vaccine production. Their metabolism was studied during growth in static (T-flasks) and dynamic cultivation systems (roller bottles, stirred tank reactor, wave bioreactor). High cell concentrations up to 5.8x10(6)cells/mL were obtained with doubling times of 23h for AGE1.CR and 35h for AGE1.CR.pIX, respectively. Both viruses were produced to high titres (3.5 logHA/100 microL for influenza virus, 3.2x10(8)pfu/mL for MVA). Hence, the CR cell lines are an appropriate substrate for pharmaceutical influenza and MVA production.

We studied vaccine strains of influenza viruses A and B during their culturing in MDCK and Vero cells grown in Eagle's MEM medium and in a medium on the basis of enzyme hydrolysate of rise flour proteins with reduced (2%) content of fetal calf serum. Optimal conditions for cell culturing and reproduction of influenza virus strains in these cells were studied. Culturing of vaccine strains of influenza viruses in MDCK and Vero cells grown in nutrient media on the basis of rise flour protein hydrolysate yielded high infection titers, which suggests that this medium can be used for the development of cultural influenza vaccine.

The recent resurgence of Chikungunya virus (CHIKV) in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, no approved licensed vaccine is currently available. In the present study, a Vero cell adapted purified formalin inactivated prototype vaccine candidate was prepared using a current Indian strain implicated with the explosive epidemic during 2006. The bulk preparation of the vaccine candidate was undertaken in microcarrier based spinner culture using cytodex-1 in virus production serum free medium. The inactivation of the virus was accomplished through standard formalin inactivation protocol. The mice were immunized subcutaneously with alhydrogel gel formulation of inactivated virus preparation. The assessment of both humoral and cell-mediated immune response was accomplished through ELISA, plaque reduction neutralization test (PRNT), microcytotoxicity assay and cytokine production assay. The results revealed that formalin inactivated vaccine candidate induced both high titered ELISA (1:51,200) and plaque reduction neutralizing antibodies (1:6400) with peak antibody titer being observed during 6 -- 8 weeks of post-vaccination. In the absence of suitable murine challenge model, the protective efficacy was established by both in vitro and in vivo neutralization tests. Further assessment of cellular immunity through in vitro stimulation of spleenocytes from immunized mice revealed augmentation of high levels of both pro- and anti-inflammatory cytokines, indicating a mixed balance of Th1 and Th2 response. These findings suggest that the formalin inactivated Chikungunya vaccine candidate reported in this study has very good immunogenic potential to neutralize the virus infectivity by augmenting both humoral and cell-mediated immune response.

Influenza virus strains of different types for use as an inactivated vaccine have been successfully grown in different cell lines. Increasing titres were obtained with BHK-21/BRS, VERO and MDCK cells. Cultures in stationary flasks, in spinner cultures or in large bioreactor systems were tested and the optimal conditions were studied. MDCK cells grown in serum-free medium before and during the virus production phase were found to yield high titres in the presence of trypsin. Satisfactory results were obtained with egg-adapted strains of human and equine origin as well as with strains just isolated from human patients without any further passages in eggs or cell culture.

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2):* BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (mu_max = 0.0510+/-0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (mu_max = 0.0305+/-0.0177 h-1).* Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively.* For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained.The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed.

To identify the molecular determinants contributing to the inability of recent human influenza A(H3N2) viruses to agglutinate chicken erythrocytes, phenotypic revertants were selected upon passage in eggs or MDCK cells. The Leu194Ile or Val226Ile substitutions were detected in their hemagglutinin (HA) sequence concomitantly with the phenotypic reversion. Remarkably, as little as 3.5% of variants bearing a Val226Ile substitution was found to confer the ability to agglutinate chicken erythrocytes to the virus population. Hemadsorption assays following transient expression of mutated HA proteins showed that the successive Gln226 --> Leu --> Ile --> Val changes observed on natural isolates resulted in a progressive loss of the ability of the HA to bind chicken erythrocytes. The Val226Ile change maintained the preference of the HA for SAalpha2,6Gal over SAalpha2,3Gal and enhanced binding of the HA to alpha2,6Gal receptors present on chicken erythrocytes. In contrast, simultaneous Ser193Arg and Leu194Ile substitutions that were found to confer the ability to agglutinate sheep erythrocytes increased the affinity of the HA for SAalpha2,3Gal.

The amino acid sequences of the HA(1) portion of the haemagglutinin of two equine A(H3N8) influenza viruses isolated in France in 1993 and 1998 were analysed to determine their evolutionary relationship with 51 other HA(1) amino acid sequences available in databanks. Our data show that the French strain isolated in 1993 belongs to a group of phylogenetically related viruses branched on the main trunk, illustrating the main lineage of evolution of the equine-2 H3 sequences before its split into two distinct lineages in the late 1980s. By contrast, the 1998 French isolate appears to belong to the more recent 'Eurasian' lineage. These data suggest that equine-2 strains antigenically related to old prototype viruses may cocirculate with the more recent 'Eurasian' and 'American' lineages. In conclusion, it may be necessary to include both strains representative of recent equine influenza variants and an older prototype strain in the current equine influenza vaccines.

Four influenza C virus strains, isolated in France in 1991, were used as a source for a kinetic study of the enzyme O-acetylesterase (EC 3.1.1.53) related to another strain, C/JHB/1/66, considered as the reference strain. Similarities, but also differences, in their haemagglutination titres were detected. Remarkable differences were found for enzyme activity and the K(m), Vmax, and the Vmax/K(m) ratio between certain strains, as well as for their thermostability at 40 degrees C when methylumbelliferyl acetate was used as substrate. By contrast, their optimum pH, stability at different pH values, and stability at 4 degrees C over 14 days were very similar. The effect of some compounds on O-acetylesterase activity was studied. The peculiarities of these factors are discussed in relation to the functional variation of the virus.

Influenza is an infection of humans beings and many animal species. It is caused by viruses which belong to the Orthomyxoviridae family. There are three types of influenza viruses A, B and C. The type A is the most pathogenic of all. The type is determined mainly by the nature of the nucleoprotein (NP), an antigen which does not greatly vary. On the contrary, the surface antigens, among which the haemagglutinin is the most important, are highly variable and their nature determines the sub-type of virus within the type A. The expressed mutations affecting the haemagglutinin are referred as antigenic drift and make virological surveillance necessary in order to annually assess the composition of the vaccine strains. The segmented nature of the genome of influenza viruses, makes possible the genetic reassortment of two different influenza viruses co-infecting one cell and produces a new hybrid virus. When such an event affects the haemagglutinin, the reassortment leads to an antigenic shift. In nature, it most certainly takes place in swine, between human and avian viruses. Whereas antigenic drift is a continuous and progressive phenomenon, antigenic shift occurs occasionally every 10 to 30 years. The emergence of a hybrid virus bearing a new haemagglutinin and thus belonging to a new human subtype, can be the starting point of the genesis of a pandemic, generally associated with a high mortality rate in humans. The participation of the pig is specially mentioned.

In order to provide an easy and powerful analysis of influenza C viral HEF RNA segment of a recent strain, a combination of reverse transcription and the polymerase chain reaction was used. We amplified the entire coding region of the HEF gene of a laboratory strain of virus called C/Johannesburg/1/66, widely used for binding and esterase activity studies as well as that of a strain isolated in 1991 (C/Paris/145/91) from a patient suffering from severe flu syndrome. The sequences we amplified were about 2 kilobases long. In this work, we show that the forward 'universal primer' Uni1, which has been used for influenza A and B viruses cDNA syntheses can also be used for influenza C virus. The PCR primers were designed to contain restriction sites to make the PCR products ready to be used for further purposes. A restriction analysis of the PCR products combined with analyses of all the human influenza C virus HEF gene sequences published so far permitted the design of sets of oligonucleotides which can prime PCR on cDNA of unknown influenza C virus for cloning.

Two seroepidemiological surveys carried out so far, one in Japan, the other in France, gave a strong indication that dogs may be naturally infected by influenza C virus, considered to be exclusively human until recently. In this work, 101 serum samples were collected during winter 1989/1990 from dogs in Castilla y León, Spain. Sera were tested for the presence of antibodies to influenza C virus by Hemagglutination Inhibition (HI) test. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. Significant HI activity was found in 56.3% of the 101 tested sera and titres ranged from 25 to 200.

From an overall point of view, the epidemiological situation of influenza C virus infections in western Europe is hardly known. In some countries like Spain, no epidemiological survey has been carried out to determine whether influenza C virus does or does not circulate and cause infection in the considered geographical area. We thus decided to perform such a study. A total of 191 serum samples was collected from people (from 1.5 to 80 years old) living in Spain in October 1990. These sera were tested for the presence of antibodies to influenza C virus by hemagglutination-inhibition (HI) tests. Significant HI activity was found in 59.3 to 64.9% of the 191 tested sera and titres ranged from 20 to 320. The high prevalence of antibody as well as the highly significant titres indicate an intense circulation of influenza C virus in Spain. A significant difference was found between children/teenagers and adults.

Influenza C virus (strain C/Johannesburg/1/66) was grown, harvested, purified and used as source for the enzyme O-acetylesterase (N-acyl-O-acetylneuraminate O-acetylhydrolase; EC 3.1.1.53). This activity was studied and characterized with regard to some new substrates. The pH optimum of the enzyme is around 7.6, its stability at different pH values shows a result similar to that of the pH optimum, and its activity is well maintained in the pH range from 7.0 to 8.5 (all these tests were performed with 4-nitrophenyl acetate as substrate). Remarkable differences were found in the values of both Km and Vmax, with the synthetic substrates 4-nitrophenyl acetate, 2-nitrophenyl acetate, 4-methylumbelliferyl acetate, 1-naphthyl acetate and fluorescein diacetate. The use of 4-nitrophenyl acetate, 4-methylumbelliferyl acetate or 1-naphthyl acetate as substrate seems to be convenient for routine work, but it is better to carry out the measurements in parallel with those on bovine submandibular gland mucin (the latter is a natural and commercially available substrate). It was found that 4-acetoxybenzoic acid, as well as the methyl ester of 2-acetoxybenzoic acid, but not 2-acetoxybenzoic acid itself, are cleaved by this enzyme. Triacetin, di-O-acetyladenosine, tri-O-acetyladenosine, and di-O-acetyl-N-acetyladenosine phosphate, hitherto unreported as substrates for this viral esterase, are hydrolysed at different rates by this enzyme. We conclude that the O-acetylesterase from influenza C virus has a broad specificity towards both synthetic and natural non-sialic acid-containing substrates. Zn2+, Mn2+ and Pb2+(as their chloride salts), N-acetylneuraminic acid, 4-methyl-umbelliferone and 2-acetoxybenzoic acid (acetylsalicylic acid) did not act as inhibitors.

ABSTRACT: BACKGROUND: Current influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines. RESULTS: We evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1x10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4x106 cells/mL can be infected without a cell density effect achieving titers of 1x108 virions/mL after 24 h. CONCLUSIONS: Overall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required.

A process for human influenza H1N1 virus vaccine production from Madin-Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional-integral-derivative control of pH, dissolved O(2)(DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0 × 10(9) to 3.2 × 10(10) cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8 × 10(7) 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1)(rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.

BACKGROUND Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1) gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. RESULTS Five commercially available animal-component free, serum-free media (SFM) were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. CONCLUSIONS We describe for the first time the production of Influenza viruses using Vero cells in commercially available animal-component free, serum-free medium. This work can be used as a basis for efficient production of attenuated as well as wild type Influenza virus for research and vaccine production.

Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines. We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration. The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2(2) full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels. On the basis of software analysis, a combination of m.o.i. of 0.0066TCID(50%)/cell and trypsin concentration of 5μg/1.0×10(6) cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells. Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium.

A downstream processing was examined for Vero cell-derived human influenza virus (H1N1) grown in serum free medium. Vero cell banks were established in serum free medium and characterized according to regulatory requirements. Serum free Vero cells were grown on Cytodex 3 microcarriers in 5L bioreactor and infected with influenza A virus (A/New Caledonia/99/55). The harvests were processed with the sequence of inactivation, clarification, anion exchange chromatography (DEAE FF), Cellufine Sulfate Chromatography (CSC) and size exclusion chromatography (Sepharose 6FF). Host cell DNA (hcDNA) was mainly removed with DEAE FF column and CSC by 40 and 223 fold, respectively. Most of Vero cell proteins were eliminated in CSC and Sepharose 6FF unit operation by about 13 fold. The overall scheme resulted in high recovery of hemagglutinin (HA) activity and the substantial removal of total protein, host protein and DNA. The total protein content and DNA content per 15 μg HA protein in final product was 89 μg and 33 pg, respectively, which complied with regulatory requirements for single strain influenza vaccines. SDS-PAGE analysis and Western blotting confirmed the purity of the final product. In conclusion, the suggested downstream process is suitable for the purification of microcarrier-based cell-derived influenza vaccine.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This problem is compounded by the fact that the pandemic-driven situation in 2009 has roughly doubled the overall vaccine demand. Modern cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the increasing worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumors have fomented uncertainty among the general population and physicians, and also discredit officially accepted test results and product licensing. This article provides an overview of the safety profile of the cell culture technology, of the cells and of the final vaccine product.

At present, the seed viruses for the manufacture of licensed seasonal inactivated influenza vaccines in the United States are derived from primary egg isolates as a result of concerns associated with adventitious agents. According to the prevailing view, the passage of influenza viruses through eggs serves as a filtering step to remove potential contaminating viruses. We have investigated the feasibility of addressing adventitious-agent risk by subjecting influenza virus to a plaque-purification procedure using MDCK cells. SV40 and canine adenovirus-1 (representing viruses for which MDCK cells are non-permissive and permissive, respectively) were used as challenge viruses to model agents of concern that might be co-isolated along with the influenza virus. By mixing influenza virus strain A/PR/8/34 with varying amounts of each challenge virus and then performing a plaque assay for influenza virus using MDCK cells, we have attempted to determine the efficiency by which the challenge virus is removed. Our data suggest that substantial removal can be achieved even after a single round of plaque purification. If cell-derived isolates were deemed to be acceptable following a plaque-purification procedure, the manufacture of seasonal influenza vaccine would be facilitated by:(1) the expansion of the repertoire of viruses from which seed virus candidates could be generated for licensed egg-derived vaccines as well as for vaccines manufactured in mammalian cells; and (2) the mitigation of adventitious-agent risk associated with the seed virus, and hence the elimination of the need to passage seed viruses in eggs for vaccines manufactured in mammalian cells.

An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.

The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3(**)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45%(v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.