Serum-free microcarrier based production of replication deficient influenza vaccine candidate virus lacking NS1 using Vero cells - PubMed (original) (raw)

Comparative Study

Serum-free microcarrier based production of replication deficient influenza vaccine candidate virus lacking NS1 using Vero cells

Allen Chen et al. BMC Biotechnol. 2011.

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 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.

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Figures

Figure 1

Vero cell microcarrier cultivation in different commercial serum-free media. (A) Comparison of Vero cell growth on Cytodex 1 microcarriers in five different serum-free media, OptiPro SFM, EX-CELL Vero SFM, VP-SFM, Provero-1 and HyQ SFM4MegaVir. The experiment was performed in duplicate using 250 ml spinner flasks with 3 g/l Cytodex 1 microcarriers. The viable cell concentrations of one representative run are shown here. (B) Phase contrast images of cells cultured on Cytodex 1 microcarriers in EX-CELL Vero SFM and OptiPro SFM at day 8. Scale bars indicate 200 μm.

Figure 2

Vero cell growth on microcarriers in OptiPro SFM with two medium exchanges. The experiment was performed in duplicate using 250 ml spinner flasks with 3 g/l Cytodex 1 microcarriers. (A) Cell growth profile and (B) Metabolite profiles of glucose, lactate, glutamine and ammonium.

Figure 3

Cultivation of Vero cells on microcarriers in a 3 L stirred tank bioreactor using EX-CELL Vero SFM with 3 g/l Cytodex 1. The experiment was performed in triplicate. (A) Cell growth profile and (B) Metabolite profiles of glucose, lactate, glutamine and ammonium.

Figure 4

Establishing trypsin concentration and MOI for ΔNS1 H1N1 Influenza virus production in Vero cells cultured on microcarriers with EX-CELL Vero SFM. Vero cells were first cultivated using 250 ml spinner flasks with 3 g/l Cytodex 1 microcarriers. Upon confluency, 80% of culture medium was replaced with fresh medium. 5 ml of this culture was transferred to each well on suspension 6-well plates to test the different trypsin concentrations and MOI for infection. Trypsin concentrations used were 10 μg/ml, 5 μg/ml, 3 μg/ml, 2 μg/ml/day and 1 μg/ml/day. Virus titres obtained using TCID50 (Top) and haemagglutination assays (Bottom) at (A) MOI of 0.001 and (B) MOI of 0.01. Virus titres shown represent mean values obtained from two replicate wells. Error bars indicate the standard deviation of the experiment.

Figure 5

Establishing time-point of infection (TOI) for ΔNS1 H1N1 Influenza virus production in Vero cells cultured on microcarriers with EX-CELL Vero SFM. (A) Growth curve of Vero cells cultivated using 250 ml spinner flasks with 3 g/l Cytodex 1 microcarriers. Cells were infected at day 5, day 6 and day 7 as depicted, by transferring 5 ml of the culture to each well on suspension 6-well plates. (B) Virus titre was measured at 48 hours post-infection for all TOI. Each bar represents the mean of the virus titres yielded from two runs using MOI of 0.001. The error bars indicate the standard deviation of the two runs.

Figure 6

Cell cultivation and ΔNS1 H1N1 Influenza virus amplification in 3 L stirred tank bioreactor using EX-CELL Vero SFM with 3 g/l Cytodex 1, MOI of 0.001 and trypsin concentration of 10 μg/ml. (A) Cell growth profile and virus amplification of representative bioreactor run. Error bars indicate the standard error of triplicate measurements. (B) Metabolite profiles of glucose, lactate, glutamine and ammonium, before and after infection. (C) Comparison of max virus titres between infection at M.O.I 0.001 and 0.01 carried out in 3 L bioreactor scale. Log2 HAU is represented as filled bars and non-filled bar indicates virus titre in Log10 TCID50/ml. Error bars indicate the standard deviation of triplicate bioreactor runs.

Figure 7

Cell cultivation and wild type H1N1 Influenza virus amplification in 3 L stirred tank bioreactor using EX-CELL Vero SFM with 3 g/l Cytodex 1, MOI of 0.001 and trypsin concentration of 10 μg/ml. (A) Cell growth profile and virus amplification of representative bioreactor run. Error bars indicate the standard error of triplicate measurements. (B) Metabolite profiles of glucose, lactate, glutamine and ammonium, before and after infection.

Figure 8

Phase contrast images of cells cultured on Cytodex 1 microcarriers after infection with wild type and ΔNS1 H1N1 Influenza viruses in EX-CELL Vero SFM. Scale bars indicate 200 μm.

Figure 9

Comparison of virus titres between ΔNS1 and the wild type H1N1 Influenza virus in EX-CELL Vero SFM and OptiPro SFM from 3 L stirred tank bioreactors. Error bars indicate the standard deviation from at least two separate runs. Log2 HAU is represented as filled bars and non-filled bar indicates virus titre in Log10 TCID50/ml.

References

1. Poland GA, Jacobson RM, Targonski PV. Avian and pandemic influenza: An overview. Vaccine. 2007;25:3057–3061. doi: 10.1016/j.vaccine.2007.01.050. -DOI -PubMed
1. Kendal AP, Maassab HF, Alexandrova GI, Ghendon YZ. Development of cold-adapted recombinant live, attenuated influenza A vaccines in the U.S.A. and U.S.S.R. Antivir Res. 1982;1(6):339–365. doi: 10.1016/0166-3542(82)90034-1. -DOI
1. ADIS. Influenza Virus Vaccine Live Intranasal - MedImmune Vaccines: CAIV-T, Influenza Vaccine Live Intranasal. Drugs R D. 2003;4(5):312–319. doi: 10.2165/00126839-200304050-00007. -DOI -PubMed
1. Maassab HF, Bryant ML. The development of live attenuated cold-adapted influenza virus vaccine for humans. Rev Med Virol. 1999;9:237–244. doi: 10.1002/(SICI)1099-1654(199910/12)9:4<237::AID-RMV252>3.0.CO;2-G. -DOI -PubMed
1. Belshe R, Lee M-S, Walker RE, Stoddard J, Mendelman PM. Safety, immunogenicity and efficacy of intranasal, live attenuated influenza vaccine. Expert Rev Vaccines. 2004;3(6):643–654. doi: 10.1586/14760584.3.6.643. -DOI -PubMed

Serum-free microcarrier based production of replication deficient influenza vaccine candidate virus lacking NS1 using Vero cells - PubMed (original) (raw)