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

Allen Chen,Say Kong Ng,Christian Dietzsch,Mylene L Yan,Swan Li Poh,Elisabeth Roethl

doi:10.1186/1472-6750-11-81

Abstract

BackgroundInfluenza 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.ResultsFive 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.ConclusionsWe 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.

Highlights

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
The live attenuated virus vaccine has been used in Russia since the 1960s [2], concerns regarding safety and possible virus shedding have precluded it from use in the rest of the world until recently: In 2003, a cold adapted, egg grown, live attenuated influenza virus vaccine by MedImmune was licensed for use in the US [3,4]
One promising live attenuated Influenza that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1) gene developed by Avir Green Hills Biotechnology [8,9,10,11,12]

Summary

Introduction

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. Responsible for millions of deaths in pandemics, the virus causes seasonal outbreaks during colder months in temperate regions which annually result in up to 500,000 Both inactivated vaccine and the live attenuated Influenza vaccines are commercially available. As NS1 is an interferon antagonist [13], the NS1 deletion virus is replication defective in interferon competent host systems, enabling its use as a live attenuated vaccine [9,10,11] Another consequence of this gene deletion is that this virus vaccine can only propagate effectively in cells with a deficiency in the interferonmediated antiviral response [8]. This NS1 deletion virus is interesting because it may find applications in cancer therapy [18,19] and other prophylactics [20]

Methods

Results

Conclusion