Attenuated hepatitis A virus
AssigneeMerck & Co., Inc.
InventorsPaula A. Giesa, Maurice R. Hilleman, Philip J. Provost
Life of this patent
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Abstract
Hepatitis A virus is attenuated by propagation in cell culture in vitro by direct inoculation of the cell cultures with a human clinical specimen containing the virus.
Claims29
A method of attenuating the virulence of hepatitis A virus comprising passaging the virus at least once in a tissue culture in which hepatitis A virus is capable of replicating using as inoculum a human clinical specimen containing hepatitis A virus.
A process for propagating human hepatitis A virus in vitro in cell culture comprising inoculating a cell culture in which hepatitis A virus is capable of replicating using as inoculum a human clinical specimen containing hepatitis A virus but in which clinical specimen hepatitis A virus is not capable of replicating, incubating the susceptible cell culture until the presence of hepatitis A virus is detected in the susceptible cell culture, and harvesting the hepatitis A virus from the cell culture.
In the method for direct isolation of HAV taken from stool samples of humans with acute hepatitis A, and further isolating and propagating the virus in a suitable substrate, the step which comprises directly passaging said virus in African Green Monkey kidney culture cells to form a serological test or radioimmunoassay of anti-HAV.
A process which comprises culturing unmodified HAV on human kidney cells (HKC) and harvesting the thus-produced viruses.
Modified HAV adapted to HKC (HAV/HKC) and having a rate of growth and/or a rate of proliferation significantly greater than that of unmodified HAV cultured on HKC.
A process which comprises:
- (a) culturing unmodified HAV on human kidney cells (HKC) and harvesting thus-produced viruses,
- (b) culturing isolated viruses from step (a) and HKC and isolating thus-produced viruses,
- (c) culturing isolated viruses from step (b) on HKC and isolating thus-produced viruses,
- (d) repeating step (c) a number of times, each with isolated viruses from the immediately preceding culturing on HKC to obtain HAV adapted to HKC (HAV/HKC) and having a rate of growth and/or a rate of proliferation significantly greater than that of HAV cultured on HKC, the isolated viruses cultured in and isolated from the products of steps (b) through (d) being those which grow rapidly and/or are produced in largest quantities;
- (e) culturing HAV/HKC isolated from step (d) on human fibroblast strains (HFS) and isolating thus-produced viruses,
- (f) culturing isolated viruses from step (e) on HFS and isolating thus-produced viruses,
- (g) culturing isolated viruses from step (f) on HFS and isolating thus-produced viruses,
- (h) repeating step (g) a number of times, each with isolated viruses from the immediately preceding culturing on HFS to obtain HAV adapted to HFS (HAV/HFS) and having a rate of growth and/or a rate of proliferation significantly greater than that of HAV/HKC cultured on HFS,
- the isolated viruses cultured in and isolated from the products of steps (e) through (h) being those which grow most rapidly and/or are produced in largest quantities.
Dependencies are inferred from the claim text.