Anne F. Pihl[1,2], Anna Offersgaard[1,2], Garazi P. Alzua[1,2], Christian K. Mathiesen[1,2], Tanja B. Jensen[1,2], Ulrik Fahnøe[1,2], Jannick Prentoe[1,2], Jan P. Christensen[2], Jens Bukh[1,2], Judith M. Gottwein[1,2]

Affiliates

[1] Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Denmark [2] Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

Abstract

Background: There is no vaccine against the hepatitis C virus (HCV), a pathogen causing chronic liver disease and at least 400,000 deaths per year. Recently established HCV cell culture systems might enable the development of a whole particle HCV vaccine.
Methods: A highly efficient JFH1-based HCV recombinant with genotype 5a (isolate SA13) specific Core-NS2 (SA13/JFH1Core-NS5B) [1] was used to establish a bioprocess allowing immunogenicity studies in mice (Fig. 1). HCV was produced in Huh7.5 cell monolayer cultures in 10-layer cell factories under serum-free conditions [2]. HCV was purified from 8-80 L supernatant by cross flow filtration, iodixanol ultracentrifugation and sephadex chromatography. BALB/c mice were immunized 4 times with UV-inactivated HCV equivalent to 7.0-8.1 log10 focus forming units (FFU) formulated with Alum+monophosphoryl lipid A or Addavax. Purified serum IgG was tested in an in vitro neutralization assay [4].
Results: Production of HCV genotype 5a in cell factories allowed for 5 harvests of 800 mL supernatant with ~6.0 log10 FFU/mL. Purification of up to 80 L culture supernatant yielded up to 2000-fold concentrated highly-pure HCV antigen with up to 25 % overall recovery. Immunogenicity testing resulted in induction of neutralizing antibodies against a homologous genotype 5a recombinant virus without hypervariable region 1 (SA13/JFH1dHVR1) [3]; as well as SA13/JFH1Core-NS5B, used for the immunizations (Fig. 2).
Conclusions: We have established a bioprocess for generation of an inactivated HCV particle vaccine antigen and obtained proof-of-concept for its immunogenicity in formulation with adjuvants licensed for human use. Future optimization aims at increasing HCV yield by establishing HCV production in high-cell-density cultures to facilitate development of a vaccine for human use.
1. Mathiesen et al., Journal of Virology,2015.89:7758-7775
2. Mathiesen et al., Virology, 2014.458-459:190-208
3. Prentoe et al., Journal of Virology,2011.85:2224-2234
4. Gottwein et al., Hepatology,2009.49:364-377