Development of methods for production of a whole virus vaccine candidate stock and novel adaptive mutations in hepatitis C virus

A method of obtaining a whole hepatitis C virus (HCV) vaccine candidate stock, the method comprising:

• a) providing a cell culture supernatant comprising HCV particles, wherein the cell culture is grown in an adenovirus expression medium, optionally supplemented with penicillin 100 U/mL and streptomycin 100 μg/mL,
• b) purifying or increasing the concentration of the HCV particles in the cell culture supernatant using cross-flow filtration,
• c) purifying or increasing the concentration of the product of step using 2-6 cushion iodixanol ultracentrifugations,
• d) collecting 2-8 fractions from step c), and selecting the fractions, which contain the most HCV particles,
• e) purifying or increasing the concentration of the selected fractions from step d) using ultracentrifugation pelleting or small-scale cross-flow filtration,
• f) purifying or increasing the concentration of the product of step e) using iodixanol gradient ultracentrifugation,
• g) collecting 10-35 fractions from the product of step f), and selecting the 2-7 fractions, which contain the most HCV particles,
• h) purifying or increasing the concentration of the selected 2-7 fractions from step g) using sephadex chromatography, and
• i) obtaining the whole HCV vaccine candidate stock.