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  • 1
    Language: English
    In: BMC Biotechnology, August 11, 2011, Vol.11, p.81
    Description: 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 x 10^6 cells/ml, whereas other SFM achieved about 1.2 x 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 Log.sub.10 TCID.sub.50 /ml was achieved using trypsin concentration of 10 [mu]g/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 x 10^6 cells/ml and virus titre of 8.3 Log.sub.10 TCID.sub.50 /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 Log.sub.10 TCID.sub.50 /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.
    Keywords: Influenza Viruses -- Health Aspects ; Influenza Vaccines -- Production Processes ; Influenza Vaccines -- Health Aspects ; Virus Replication -- Health Aspects ; Swine Influenza -- Health Aspects ; Trypsin -- Health Aspects
    ISSN: 1472-6750
    Source: Cengage Learning, Inc.
    Library Location Call Number Volume/Issue/Year Availability
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  • 2
    Language: English
    In: BMC Biotechnology, August 11, 2011, Vol.11, p.81
    Description: 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 x 10^6 cells/ml, whereas other SFM achieved about 1.2 x 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 Log.sub.10 TCID.sub.50 /ml was achieved using trypsin concentration of 10 [mu]g/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 x 10^6 cells/ml and virus titre of 8.3 Log.sub.10 TCID.sub.50 /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 Log.sub.10 TCID.sub.50 /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.
    Keywords: Influenza Viruses -- Health Aspects ; Influenza Viruses -- Physiological Aspects ; Influenza Viruses -- Research ; Virus Replication -- Research
    ISSN: 1472-6750
    Source: Cengage Learning, Inc.
    Library Location Call Number Volume/Issue/Year Availability
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  • 3
    Language: English
    In: Journal of virology, November 2011, Vol.85(21), pp.11139-45
    Description: In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.
    Keywords: Amphotericin B -- Metabolism ; Antifungal Agents -- Metabolism ; Influenza A Virus -- Drug Effects ; Influenza B Virus -- Drug Effects ; Virus Replication -- Drug Effects
    E-ISSN: 1098-5514
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