High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

Anne F Pihl,Jens Bukh,Judith M Gottwein,Jannick Prentoe,Anna F Offersgaard,Christian K Mathiesen,Ulrik Fahnøe,Henrik Krarup

doi:10.1038/s41598-018-35010-5

Anne F Pihl, Jens Bukh + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-35010-5

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Abstract

Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log10 FFU/mL and 10.4 log10 IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.

Highlights

Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus of the Flaviviridae family[1]
hepatitis C virus (HCV) production in hollow fiber bioreactor (HFBR) was initially established under serum-containing conditions
The total amount of HCV Core accumulated during the main production phase in Adenovirus Expression Medium (AEM) was 1.0 × 108 amol, amounting to ~2.1 μg Core (Fig. 3c)

Summary

Introduction

Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus of the Flaviviridae family[1]. Individuals with HCV-induced hepatitis typically show no or unspecific symptoms, but have an increased risk of developing liver cirrhosis and hepatocellular carcinoma. To study HCV resistance to DAA and to develop a cell culture based HCV vaccine, cell culture systems are required[15]. Various infectious cell culture systems producing HCV particles of the major genotypes were developed[15]. Virus yields in monolayer culture are typically limited, while development of a whole virus HCV vaccine and other applications, such as morphological studies of HCV particles, require large amounts of viral particles. No high-yield, high cell density HCV cell culture systems for efficient production of HCV have been established. We demonstrate the use of this platform for studies of DAA

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