PROCESS FOR THE IN VITRO CULTURE OF HEPATITIS B VIRUS

Abstract: Hepatitis B virus is propagated in cell culture in vitro by direct inoculation of a susceptible animal with an infected clinical specimen from a human or non-human primate, and subsequent culturing of liver tissue from the infected animal. The cultured liver tissue produces hepatitis B virus antigens over an extended period of time.

Description: PROCESS FOR THE IN VITRO CULTURE OF HEPATITIS B VIRUS BACKGROUND OF THE INVENTION Heretofore the only source of hepatitis B surface antigen (HBsAg) has been blood or tumor cell culture from human carriers of hepatitis B disease. Such human carriers are difficult to locate with the result that the needed blood is scarce and expensive. There has been no method of cultivating hepatitis B virus in vitro. OBJECTS OF THE INVENTION It is an object of the present invention to provide a method for obtaining hepatitis B virus which does not require blood from human carriers. Another object is to provide a method for the in vitro cell culture of hepatitis B virus. These and other objects of the present invention will be apparent from the following description. SUMMARY OF THE INVENTION In vitro cell cultures derived from liver of a hepatitis B infected primate produce hepatitis B surface and core antigens over an extended period of time. DETAILED DESCRIPTION The present invention relates to the in vitro cell culture propagation of hepatitis B virus and, more particularly, to in vitro cell culture propagation of hepatitis B virus in cell cultures derived from the liver of a hepatitis B infected primate. According to the present invention a clinical specimen containing hepatitis B virus, e.g. blood, isolated virus, liver, plasma, saliva or serum from a human or non-human infected with hepatitis B, is used as the inoculum. The non-human primate may be any species susceptible to hepatitis B disease, e.g., baboon, chimpanzee, gorilla, orangutan, or rhesus monkey. The clinical specimen may be used to inoculate a non-human primate. The inoculation is preferably by intravenous injection. At from about 42 to about 90 days post inoculation, typically at about 70 days post inoculation, a liver specimen from the infected animal is used to establish an in vitro cell culture. Alternatively, a liver specimen from a human patient suffering from hepatitis B may be used. The infected liver cell culture is incubated in the presence of a suitable nutirent medium. By a nutrient medium is meant a cell culture medium which permits growth of cells in vitro. Some specific nutrient media are, for example, Medium 199, Morgan et al., Proc. Soc. Exp. Biol. & Med., 73:1-8, 1950; Basal Medium Eagle, Eagle, Science, 122, 501-504, 1955; In Vitro, Vol. 6, No. 1970; Dulbecco's Modified Eagle's Medium, Dulbecco et al., Virology, 8, 396, 1959; Smith et al, J. Viol., 12, 185-196, 1960; In Vitro, Vol. 6, No. 2, 1970; Minimum Essential Medium (Eagle), Science, 130, 432 (1959), RPMI Media, Moore et al., 199, 519-524, 1967; In vitro, Vol. 6, No. 2, 1970, Williams Medium E, Exp. Cell Res., Vol. 69, 106-112, 1971; or Ham's F12, 1965 PNAS, Vol. 53, 288; in the presence of serum, e.g., chimp serum or fetal calf serum. The incubation is preferably effected in the presence of one or more hormones which inhibit growth of fibroblasts. Such hormones are, for instance, insulin, glucagon and 9-fluoro-11B,17,21-trihydroxy- 16a-methylpregna-1,4-diene-3,20-dione. These substances are generally employed at a concentration -5 -7 of from about 105 to about 10 M. The incubation pre- ferably is carried out at from about 32 to about 39eC, most preferably at about 350C in an atmosphere of air containing about 5% CO2 by volume. A vigorous outgrowth of hepatocyte-like epithelial cells usually begins within 7 days. Nine days post planting hepatatis B core antigen (HBAg) is detected by fluorescent antibody or radioimmune assay (RIA) while HB5Ag is detected by RIA. These cultures continue to produce hepatitis B core and surface antigen for a period of at least 150 days. Optionally, but preferably, the hepatitis B virus is serially transmitted through at least one susceptible primate prior to removal of the liver specimen for in vitro cell culture. The following example:illustra.tes the present invention without, however, limiting the same thereto. EXAMPLE A chimpanzee is inoculated intravenously with 1 ml of a 1:1,000 dilution of a human hepatitis B carrier plasma. Liver tissue is removed 43 or more days following inoculation and finely minced by chopping, the mince is washed and planted in cell culture medium (Williams Medium E with 0 to 20% animal serum, 10 6M insulin, dexamethasone, and glucagon). Cultures in 25 cm flasks are refed with 2 to 3 ml of culture media 2 to 3 times per week. Incubation is at 350C on a rocker platform in a gas-atmosphere of 5% CO2 and air. Within 7 days a vigorous outgrowth of hepatocyte-like epithelial cells begins. This description of chimpanzee liver cell culture is pertinent whether the liver is obtained from normal or hepatitis B infected animals. HUB AT is detected by flourescent antibody at nine days post planting in the nuclei of hepatocyte-like cells which have grown out from the mince. It is possible to specifically block this fluorescence by convalescent human hepatitis B serum but not by pre-illness serum from the same patient, and by post-immunized but not pre-immunized guinea pig serum. HB5Ag is also detected by RIA in the culture -fluids either going from a negative to a positive or maintaining a positive state after repeated dilution due to refeeding. Uninfected chimp livers do not develop this effect. HB5Ag and HUB AT continue to be produced for at least 150 days.

Claims: WHAT IS CLAIMED IS: 1. A process for the in vitro cell culture of hepatitis B virus comprising incubating at a temperature of from about 32 to about 39"C in the presence of a nutrient medium a cell culture of liver tissue removed from a human or non-human primate infected with hepatitis B, and continuing the incubation until the presence of hepatitis B viral antigens is detected in the cell culture. 2. A process according to claim 1 wherein hepatitis B viral antigens are harvested from the cell culture. 3. A process according to claim 1 wherein the incubation takes place in the presence of a substance which inhibits growth of fibroblasts. 4. A process according to claim 3 wherein the substance is insulin, glucagon or 9-fluoro-llB,17,21- trihydroxy-16a-methyl-pregna-1,4-diene-3,20-dione. 5. A process according to claim 4 wherein the substance is present in a concentration of from about 10 5 to about 10 7 Mole. 6. A process according to claim 1 wherein the incubation takes place at from about 32 to about 390C. 7. A process according to claim 1 wherein the incubation takes place at about 350C. 8. A process according to claim 1 wherein the hepatitis B virus is serially transmitted through one or more susceptible primates prior to removal of liver tissue for cell culture. 9. A process according to claim 8 wherein hepatitis B viral antigens are harvested from the cell culture.

Inventors: HILLEMAN, MAURICE R., KELLER, PAUL M., MCALEER, WILLIAM J., PROVOST, PHILIP J.
Applicants: MERCK & CO. INC.
Application year: 1980
Main classification: A61K39
IPC codes: C12N7, A61K39, C12R1