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

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.

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine

“…In order to investigate the applicability of the developed DSP for different HCV isolates, we applied this strategy to a high-titre cell culture-derived genotype 5a virus 37 , 38 , which was selected due to its efficient growth characteristics in cell culture. Compared to the genotype 1a virus, the genotype 5a structural proteins differ by ~ 20%, while the envelope proteins differ by ~ 26% on the amino acid level.…”

“…For genotype 5a HCV, 18 × 10 6 cells, seeded the previous day in DMEM, were infected at a multiplicity of infection of 0.003 in T500 triple layer cell culture flasks with a 3rd passage seed stock of the further adapted SA13/JFH1 recombinant 37,41 . The following day, cells were transferred to cell factories (Nunc Cell Factory).…”

Three-Dimensional Cell Culture Systems for Studying Hepatitis C Virus

“…Using FLC-4 cells cultured in the radial flow bioreactor, Murakami et al [ 38 ] propagated HCV from carriers’ serum samples and showed the change in quasispecies composition. In a study of HCV production, Pihl et al [ 39 ] cultured Huh-7.5 cells in the hollow fiber bioreactor. Since the cells grew at a higher density in the bioreactor than in monolayers [ 37 ], higher titers of HCV were produced.…”

“…Since the cells grew at a higher density in the bioreactor than in monolayers [ 37 ], higher titers of HCV were produced. When the cells were treated with the NS5A inhibitor daclatasvir, lower infectious viral titers were observed [ 39 ]. However, the morphology of Huh-7.5 cells in the hollow fiber bioreactor is yet to be evaluated.…”

Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens

“…Studies have shown that mouse immunizations with inactivated particles lead to the induction of NAbs, but NAb titers in immunized animals have so far been modest [42,43]. In addition, despite recent advances in production and downstream processing of inactivated HCVcc [44][45][46][47], challenges persist in large-scale HCVcc production. Thus, the use of subunit vaccine approaches is an attractive alternative.…”