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Berlin Brandenburg

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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.
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  • 2
    Language: English
    In: PLoS ONE, 2012, Vol.7(6), p.e39921
    Description: The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.
    Keywords: Research Article ; Biology ; Medicine ; Immunology ; Virology ; Infectious Diseases ; Microbiology
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: BMC Biotechnology, 01 August 2011, Vol.11(1), p.81
    Description: Abstract 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...
    Keywords: Influenza ; Vero ; Microcarrier ; Ns1 ; Bioreactor ; Engineering
    ISSN: 1472-6750
    E-ISSN: 1472-6750
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  • 4
    Language: English
    In: The Journal of infectious diseases, 01 February 2010, Vol.201(3), pp.354-62
    Description: BACKGROUND. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-H1N1 vaccine was well tolerated. Rhinitis-like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. CONCLUSIONS. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. TRIAL REGISTRATION. ClinicalTrials.gov identifier: NCT00724997 .
    Keywords: Influenza A Virus, H1n1 Subtype -- Immunology ; Influenza Vaccines -- Immunology ; Influenza, Human -- Prevention & Control ; Vaccines, Attenuated -- Immunology ; Viral Nonstructural Proteins -- Genetics
    ISSN: 00221899
    E-ISSN: 1537-6613
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  • 5
    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.
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  • 6
    Language: English
    In: Vaccine, 01 May 2014, Vol.32(21), pp.2487-2492
    Description: We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1 virus were 1.9E + 10 TCID /ml, 1.0E + 10 TCID /ml and 8.9E + 08 TCID /ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8 ± 32.3% and 87 ± 30.8%. Total protein removal varied from 93.3 ± 0.4% to 98.6 ± 0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease.
    Keywords: Influenza Viruses ; Vaccines ; Purification ; Chromatography ; Monoliths ; Medicine ; Biology ; Veterinary Medicine ; Pharmacy, Therapeutics, & Pharmacology
    ISSN: 0264-410X
    E-ISSN: 1873-2518
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  • 7
    Language: English
    In: Journal of Virology, 2011, Vol. 85(21), p.11139
    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 -- Growth & Development; Virus Replication -- Drug Effects; Virus Replication -- Growth & Development; Virus Replication -- Drug Effects;
    ISSN: 1098-5514
    ISSN: 10985514
    ISSN: 0022538X
    Source: American Society of Microbiology
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  • 8
    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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