AU647384B2 - Non-A, non-B hepatitis hepatocyte cell culture - Google Patents

Description

OPI DATE 26/09/90 APPLN. ID 51666 PCT AOJP DATE 25/10/90 PCT NUMBER PCT/US90/00915 INTERNATIIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/10060 C12N 7/00, C12Q 1/70, 1/02 Al A01N 63/00 (43) International Publication Date: 7 September 1990 (07.09.90) (21) International Application Number: PCT/US90/00915 (74) Agents: PETERSON, Gale, R. et al.; Cox Smith Incorporated, 2000 NBC Bank Plaza, 112 East Pecan Street, (22) International Filing Date: 22 February 1990 (22.02.90) San Antonio, TX 78205-1536 (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 315,288 24 February 1989 (24.02.89) US pean patent), CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), IT (European (71) Applicant: SOUTHWEST FOUNDATION FOR BIOM- patent), JP, LU (European patent), NL (European pa- EDICAL RESEARCH [US/US]; P.O. Box 28147, San tent), SE (European patent).

Antonio, TX 78284 (US).

(72) Inventors: LANFORD, Robert, E. 1902 Hillingway, San Published Antonio, TX 78248 BURK, Kenneth, H. 15630 With international search report.

Cloud Top, San Antonio, TX 78248 JACOB, James, R. 9712 Village Briar, San Antonio, TX 78250

(US).

647384 (54) Title: NON-A, NON-B HEPATITIS HEPATOCYTE CELL CULTURE (57) Abstract A Non-A, Non-B hepatitis (NANBH) viral in vitro cell culture is discdosed. Primary hepatocytes were isolated and cultured from a chimpanzee during the acute phase of an experimental NANBH virus infection. The differentiated hepatocyte cell culture was maintained in a serum-free medium comprising Williams medium E, a hepatocyte proliferogen, transferrin, serum albumin, corticosteroide prolactin, thyrotropin-releasing factor, cholera toxin and ethanolamine. The cultured hepatocytes tested positive for the expression of a NANBH-associated cytoplasmic antigen. The presence of this cytoplasmic marker suggested persistence of the infection in vitro. The production of infectious virus in vitro was confirmed by inoculating a chimpanzee with NANBH virus-infected tissue culture and later documenting the NANBH development in the chimpanzee.

DESCRIPTION:

NON-A, NON-B HEPATITIS HEPATOCYTE CELL CULTURE FIELD OF THE INVENTION The present invention relates to an in vitro cell culture system capable of maintaining in culture Non-A, Non-B hepatitis (NANBH) virus. More particularly, the invention relates to cultured primate hepatocyte cells which have the ability to support replication of the NANBH virus in culture.

VACKIL OUL V L THELJLMElaTfNT Non-A, Non-B hepatitis has long been recognized as a .virus-induced disease, distinct from other forms of viral-associated liver diseases, including hepatitis A virus (HAV) and B virus (IIDV), and the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV).

Yet, despite years of extensive research, the NANBH virus has eluded isulatiorn, characterization and in vitro cultivation. A considerable amount of data suggests the existence of two or more types of NANBH virus. The two general types are distinguished by mode of transmission, namely parenteral and enteric. Of the two, the iparenterally transmitted form is associated with chronic hepatitis. Shorey, James, Amer. J. Med. Sci. 289:25-2G6 25 (19 A decade has passed since the first experimental transmission of the human infectious NANBH agent to a chimpanzee, the only reliable animal model for this disease. Yet, to date, no tissue culture system has been developed which would maintain NANBH virus in culture.

Consequently, the limited availability of an animal model and the absence of an in vitro tissue culture model have severely hampered the isolation and characterization of this elusive agent. Without the ability to isolate and chiarcterize the NANH virus, researchers are stymied in their attempt., Lo develop diagnostic reagents, therapeutic compound., and vaccines foi this disease. Due to the lack of a diaanostic tool or vaccine, approximately 901 of post-transfu-ion associated hepatitis can be 39 1- WO 90/10060 PCr/US90/00915 2 attributed to this putative viral agent. Some researchers have suggested that this infectious agent has properties consistent with the togavirus family. It is of interest that we have observed particles by electron microscopy in partially purified human serum containing infectious NANBH virus that have the morphology of the togavirus family.

To date, the inability to maintai.n differentiated primate hepatocyte cultures has probably been the biggest single obstacle to the isolation, characterization and in vitro culture of human infectious NANBH virus. While avian hepatocyte cell cultures capable of replicating duck hepatitis B have been reported (Tuttleman et al., J. Virol. 58:17 (1985)), these cell cultures have not been useful for the propagation of human hepadna virus.

Hepadna viruses exhibit a narrow host range: chimpanzees are the only species other than man that can be infected with human hepatitis B virus. Recently, primary human hepatocytes maintained in a medium containing dimethylsulfoxide were shown to be susceptible to exogenous infection with HBV. These cultures are short-lived, poorly differentiated, and have not been shown to be susceptible to NANBH virus. Fourel et al., J. Virol.

62:4136-4143 (1988).

Recently, a hormonally defined, serum-free differentiated primate hepatocyte cell culture medium has been developed. See U.S.S.N. 222,569, filed July 1988. Cultured differentiated primate liver cells offer many advantages for biochemical, viral culture and carcinogenesis studies. A system in which adult primate hepatocytes can be successfully cultured while maintaining differentiation of cell function and morphology offers tremendous possibilities in aiding the study of acute and chronic viral hepatitis and isolation of hepatotrophic viruses.

i- a r h M l)r~ iere bheefere a iep-c- -A-V n in vitre VrrTL xrr rlu ell cuiturc-m4-. hIU eh--e sucLain repli in i3 pv-n -f-A-I 4-s-nh Thereis, therefore, a need for cultured primate hepatocyte cells which have the ability to support replication of NANBH virus in culture. Such cultured cells may ultimately lead to the isolation and characterization of the NANBH virus and eventually lead to the diagnostic and therapeutic agents specific for combatting NANBH virus infection.

SUMMARY OF THE INVENTION Accordingly the present invention provides cultured primate hepatocyte cells derived from non-fetal primate liver and characterized by normal hepatocyte function for an extended culture period, as evidenced by: levels of apolipoprotein A-I and apolipoprotein E secretion in culture over a 100 day culture period which are similar to that found in primate plasma; levels of albumin secreted into culture over a 100 day culture period which is similar to that found in primate plasma; and ability to support replication of parenterally-transmitted-NANBH virus in culture.

There is also provided a primate hepatocyte culture containing replicating parenterally-transmitted-NANBH virus comprising: primate hepatocyte cells infected with the NANBH virus characterized by active replication of the NANBH S virus for up to 19 days following infection of the cells by the virus; and 3 0 a culture medium, which is effective to maintain normal hepatocyte cells, in the absence of viral infection, in a differentiated state for up to 100 days.

The present invention also provides a serum-free, cell-free isolate of NANBH virus. The isolate of NANBH virus is obtained as either a culture medium supernatant 39 3 or a lysate of the in vitro cell cultured NANBH virus-infected hepatocytes.

Accordingly the present invention also provides a method of providing an infective parenterally-transmitted-NANBH viral composition, comprising: obtaining hepatocyte cells infected with the NANBH virus; culturing the cells under conditions effective to maintain normal hepatocyte cells, in the absence of viral infection, in a differentiated state for up to 100 days; and producing from the cultured cells said composition.

Further, the present invention includes methods of producing NANBH virus infection in chimpanzeees, Such controlled NANBH virus infection of chimpanzees should provide an experimental model for the study of NANBH virus infection and serve as reservoir for production of antibodies to NANBH virus. The NANBH virus infection is induced by inoculating chimpanzees with an infectious amount of an inoculum comprising an in vitro culture of NANBH virus-infected hepatocytes, a cell-free supernatant of the in vitro culture, lysate of the in vitro culture, or cultured hepatocytes separated from their in vitro culture medium.

Further, the present invention provides a method of confirming NANBH viral infection in a host. The method involves excising hepatocytes from the host, culturing the hepatocytes, and observing cytopathic effects of the cultured hepatocytes after about two to four weeks. The cytopathic effects of the hepatocytes is indicative of NANBH virus infection.

S39 3a

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o WO 90/10060 PCT/US90/00915 4 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following described serum-free media exemplifies the formulations of the present invention which are useful in sustaining NANDH virus in primate hepatocyte cell culture. While the examples demonstrate in vitro culturing of NANBH virus-infected chimpanzee hepatocytes, the culturing medium and techniques should be understood to apply as well to include in vitro culturing of NANBH virus in human hepatocytes.

In the described media of Table 1, Williams Medium E (WME) served as a basal medium. Although WME is presently preferred as the basal medium of the serum-free medium of the present invention, it will be understood by those skilled in the art who have the benefit of this disclosure that other commercial media formulations can be expected to give satisfactory results. For instance, a mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (.see Salas-Prato, in Growth of Cells in Hormonally Defined Media, Book A, G. H. Sato, et al., Eds., Cold Spring Harbor Laboratory, pp. 615-624 (1982)) or RPMI 1640 (Gibco) (see Enat, et al., Proc. Natl. Aca.

Sci. USA 81:1411 (1984) and Sell, M. et al., "Longterm culture and passage of human fetal liver cells that synthesize albumin," In Vitro_Cell. Dev. BiQl. 21:216-220 (1985)) should give saLisfactory results when supplemented with the supplements listed in Table 1.

TABLE 1 SuDpplement Medium___nce n tration EGF 100 ng/ml Insulin 10 pg/ml Glucagon 4 pg/ml BSA 0.5 mg/ml Linoleic Acid 5 pig/ml -6 Hydrocortisone 10 M Selenium 10-7 M Seleniumn 2.0 N WO 90/10060 PCr/US90/00915 5 Suqpplement Medium _C_on entr a tion Cholera Toxin 2 ny/ml LGF 20 ng/ml Transferrin 5 pg/ml -6 Ethanolamine 10 M Prolactin 100 ng/ml Somatotropin 1 pg/ml -6 TRF 10-6 M Although the exact function of many of the various additives with which the basal medium is supplemented is not well-defined, several of the supplements can be grouped in provisional categories to facilitate the description of the media formulation of the present invention. For instance, the term "hepatocyte proliferogen," as used herein, refers to one or more of the several growth factors or hormones, such as epidermal growth factor (EGF), insulin, liver growth factor (LGF), and glucagon, all of which have been implicated in controlling liver growth in vivo (Salas-Prato, supra, at 615).

The term "transport protein" as used herein refers to those proteins found in the serum which include as one of their functions the transport of certain substances in the blood. Such proteins include serum albumin, which may be advantageously used in the commonly available form of bovine serum albumin (BSA), and transferrin. However, liver cells synthesize transferrin such that satisfactory hepatocyte maintenance may be achieved without the addition of that transport protein.

The trace metal specifically contemplated for use in the medium of the present invention is selenium; however, WME contains copper, zinc, cobalt and iron and either WME, or other base-l media, can be additionally supplemented with either o0 both of zinc and/or copper depending upon the original condition of the hepatocytes WO 90/10060 PC/US90/00915 6 and whether the basal medium includes either or both of those trace metal(s).

The literature reports the use of several growth and/or releasing factors which have been used for culturing liver cells, including thyrotropin-releasing factor (TRF), fibroblast growth factor, platelet-derived growth factor, multiplaction-stimulating activator, and endothelial cell growth supplement (ECGS) (for a review, see Leffert, H. L. and K. S. Koch, "Hlepatocyte growth regulation by hormones in chemically defined media: A two-signal hypothesis," _in Growth of Cells in Hormonally Defined Media, Book A, G. H. Sato, et E Cold Spring Laboratory, pp. 597-613 (1982)). Any one or more of those growth/releasing factors can be added to the media of the present invention, depending upon factors such as the original condition of the hepatocytes and the particular protocol to be utilized.

Although the supplements are set out in specific proportions in the following table, it will be understood by those skilled in the art who have the benefit of this disclosure that those proportions can be, and in some circumstances, must be, varied. For instance, ECGS has been found not to be required for maintenance of the hepatocytes. The concentration of glucagon in the media can be reduced. Also, there is some interchangeability between certain of the supplements. For instance, the addition of soybean lipids may be substituted for linoleic acid. In addition, the quality of the hepatocytes obtained from different isolations may require the use of different hepatocyte proliferogens.

The media of the present invention, therefore, includes a range of proportions of each of the supplements as shown in Table 2.

WO 90/10060 WO 9010060PCT/IJS9O/0091 -7- TABLE 2 Supplement

EGF

Insulin Glu ca go n

BSA

Soybean lipids Linoleic acid Hydrocorti sone Selenium Cholera toxin

LGF

EGGS

Transferrin E than ol1ain ne Prolactin Somatotropin

TPF

>25 ng/iml S2 iig/rnil 20.5 pg/inl )0.2 mg/nfl 0-20 pgq/ml 0-5 pg/ml 9 10 14 0-5 ng/ml 0-50 ng/ml 0-60 p-g/rnl 0-10 pg/rnl 10- M 0-200 ny/ml 0-5 jtg/mi 6 M Preferred Range 50-100 ng/rnl 5-10 pg/ml 0.5-10 pg/ml 0.5-2 mg/ml 0-20 p.g/ml 0-5 vtg/ml 10- 10- 6 3 x 10- 10- 7 0-2 ng/ml 0-20 ng/ml 0-60 Pg/MI 0-5 Vig/ml 10- 6 14 100 ng/ml 1 Pg/ml 0-1.0- M It will be further understood that, with respect to the proportions of each of those supplements, when it is stated that a media formulation includes, for instance, 10- G M TRE, the media includes ab~out 10-6 M TRF.

The several studies that were performed to evaluate the ability of the media formulation of Example 1 of the present invention to support NANBH viral replication will now be described. The isolation of NANBH virus-infected hepatocytes from chimpanzees is described in Examples 7.

and 4.

ExAmrLE I SeLrm-F iee M~iunFtq~jLaUjQU The serum-free meli.i f ormula Lion utilized WME as a basal medium suppletnented with 10 mM HEPES, pH 7.4, 2.75 mg/ml NaHCO V an11d 50 Pg/ml gentamycin. TO WO 90/10060 PCT/US90/00915 8 prepare the media, the supplements were added in the following quantities to 500 ml of WME in a sterile plastic bottle: ml 50 mg/ml BSA, 500 pg/ml Linoleic Acid 0.5 ml 5 mg/ml Insulin ml 5 mg/ml Insulin, 5 mg/ml Transferrin, and pg/ml Selenium (ITS) pi 10 2 M Hydrocortisone p1 200 pg/ml Cholera toxin 0.5 ml 100 pg/ml EGF -2 p1 10 2 M Ethanolamine ml 1 ig/ml Somatotropin pi 1 mg/ml Prolactin -3 ml 10 3 M Tlyrotropin Releasing Hormone 50 pl 200 pg/ml LGF 1 ml 2.0 mg/ml Glucagon WME was purchased with L-glutamine and without NaHCO 3 from Hazelton Research Products, Inc. (Denver, Penn.).

EXAMPLE 2 Chimpanzee Experimental NANBH Infection In order to obtain NANBH virus-infected hepatocytes for in vitro experimentation, a parenteral NANBH virus infection was induced in chimpanzee PTTx7, a 14-year-old female, by inoculation with 5 ml of a 20-fold concentrate of acute phase plasma of unknown titer detrved from a second passage of the Hutchinson strain of NANBH virus.

Progression of the NANDI virus infection was monitored by ALT/AST enzyme fluctuations from weekly blood samples and by histopathologic examination of periodic liver needle punch biopsies. All biopsies were processed identically using conventional techniques. Immediately after harvesting, the liver biopsies were fixed for 1-3 hours in neutral buffered 3.7% formalin, processed manually according to standard procedures, embedded in paraffin, sectioned at 4 microns and stained with hematoxylin and eosin. All sections were examined histologically by the same board certified veterinary pathologist.

WO 90/10060 PCT/US90/0091 9 Since the onset of clinical hepatitis was significantly delayed, a second inoculation of 1.5 ml (102.5 CID 5 0 NANBH vi.rus Hutchinson inoculum was administered on week 10 to assure infection. However, the appearance of elevated ALT on week 12 indicated that the second inoculum either exacerbated the primary infection or was not required. The ALT profile of the animal exhibited a rise above normal values from 12-19 weeks post-inoculation, and a second ALT elevation occurred on week 39.

Liver wedge survery was performed on week 14 at the onset of definitive ALT elevation. Microscopic examination of liver tissue taken at this time revealed occasional collections of lymphocytes and macrophages in hepatic triads and in focal parenchymal areas. There were no other changes indicating a significant inflammatory response. Although minimal inflammation was present, this finding could be representative of normal liver tissue. Ilepatocytes were isolated on week 14 under the pretense that maximal virus replication would occur prior to or during this stage of the disease manifestation.

A liver punch biopsy taken after ALT elevations (week 19) revealed an increased number of lymphocytes in portal areas and in the parenchyma of the liver. Associated with the parenchymal lesions were necrotic hepatocytes.

The hepatocytes around central vein areas were often lightly stained and granular with minimal swelling of the cytoplasm. All these changes described indicated minimal, lymphocytic, multifocal, viral hepatitis.

Development of In Vitro NANHII Virus- S.nfected He picyLe Ce__llCul___ure__.

Ketamine hydrochloride was used as the immobilizing and pre-anesthetic agent. Surgery was performed under general anesthesia with non-hepatotoxic sodium pentobarbital. A liver wedge of approximately 10 g was perfused using a modification of established protocols NO 90/10060 pC/US90/00915 10 (Maslansky, C. J. and G. M. Williams, In Vitro Models for Cancer Research, Vol. II: Carcinomas of the LIver and Pancreas, M. M. Weber and L. I. Sekely CRC Press: Boca Raton, Fla., pp. 43-60 (1985)). A two-step perfusion procedure was employed with all solutions maintained at 37 0 C throughout the perfusion procedure.

The initial perfusion lasted 10 minutes using 1 liter of Ca Mg -free Hanks Balanced salt sclution supplemented with 10 mM HEPES (pH 0.5 mM EGTA, and 100 ig/ml gentamycin sulfate. The next perfusion was for 20 minutes at approximately 60 ml/min. of Williams Medium E (WME) supplemented with 10 mM IIEPES 'pll 7.4), 100 pg/ml gentamycin sulfate, and 200 units/ml collagenase Type I (300 units/mg, Sigma). The liver capsule was then removed with fine forceps and hepatocytes were dislodged by gentle agitation in 100 ml of coliagenase solution. The hepatocyte suspension was filtered through several layers of gauze pads into an equal volume of cold WME containing 5% fetal bovine serum (FBS), 10 mM {HEPES (pH and 100 pg/ml gentamycin sulfate. Hepatocytes were dimented at 50 x g for minutes and cell pellets were resuspended in WME FDS. Sedimentation was repeated twice, pellets were -esuspended in 10 ml WME 5% FDS, and viability and cell density were determined by trypan blue exclusion.

PRIMARIA plates (Falcon) were coated with rat tail collagen (Michalopoulos, G. and H. C. Pitot, "Primary culture of parenchymal liver collagen membranes," Exptl.

Cell. Res. 94: 70 (1975)) for 6 minutes at room temperature, the excess collagen was removed, and plates were dried oveLnight under U.V. light. Viable cells were plated at a density of 3-4 x 106 cells/60 mm dish Cell attachment occurred during a 3-ho-r incubation at 37 0 C, 10% CO 2 in WME 5% FDS, at which time cell monolayers were gently washed one time with WME and re-fed with serum-free medium formulation prepared as described in Example 1 above. The medium was changed PC/US90/00915 WO 90/10060 11 24 hours after isolation and at 48-hour intervals thereafter.

The cultured hepatocytes displayed a typical hepatocyte morphology as observed by phase-contract microscopy on day 5 of culture. This morphology was maintained until days 21-28 when the cultures exhibited a degenerative process.

NANBH Hepatocvte Cell Culture Characteristjic The synthesis and secretion of albumin, apolipoprotein A-I, and apolipoprotein E were monitored by immunoblottinq of aliquots of tissue culture medium.

Briefly, proteins were separated by sodium dodecyl sLlfate-polyacrylamide gel electrophoresis (SDS-PAGE,) and "-ere electrophoretically transferred to Nylon-X nitrocellulose filters (Fisher) at 100 mA for 16 hours at 4°C. Unoccupied binding sites were blocked in 10% nonfat dry milk in phospate buffered saline (PBS) for 2 hours at 37 0 C. Membranes were incubated for 2 hours at 37 0 C in PBS-milk-Tween (PBS containing 5% nonfat dry milk, 0.3% using primary antibodies directed against human apolipoproteins A-I and E. Membranes were washed three times with PBS-Tween and incubated 1 hour at 37°C in PBS-milk-Tween with antibodies directed against each of the primary antibodies. Membranes were washed three times with PBS-Tween and incubated 1 hour at 37 0 C in PBS-milk-Tween with [125 protein A (8.5 PCi/pg, NEN). Membranes were washed three times with PBS-Tween and air dried. Immunoblots were autoradiographed at -85 0 C on XAR-5 film (Kodak) with intensifying screens.

The levels of apolipoproteins A-I and E increased in the cultures up to dgy 13, remained constant from day 13-28, and declined from day 28-45.

Albumin detected by this immuroblot procedure remained at constant levels throughout the culture period. Although albumin is a marker for differentiated hepatocytes, it is not as stringent of a marker for the differentiated state as is lipoprotein synthesis.

WOr 90/10060 P@c/US90/00915 12 The decline in lipoprotein synthesis after 28 days in culture paralleled a degeneration in the hepatocyte cultures. The degeneration of primary hepatocytes after 3-4 weeks of culture was evident in cultures derived from two different NANBH-infected chimpanzees, but was not observed in cultures from a normal chimpanzee or chimpanzees with HBV infections. Normal hepatocyte cultures generally survive more than 100 days in the serum-free media. Further experimentation will be required to determine whether the degenerative process is due to viral-induced cytopathic effect.

To further characterize the differentiated state of the hepatocytes in vitro, the de novo synthesis of liver specific plasma proteins was analyzed. On day 17, cul- Lures were labeled for 24 hours with Plasma proteins were immune precipitated from the labeled medium and analyzed by SDS-PAGE.

Cultures were incubated in 2.5 ml of the serum-free media of Example 1 supplemented with 250 pCi 35Smethionine (>800 Ci/mmol, ICN) for 24 hours.

Medium was filtered and mixed with 1/10 volume of CHAPS extraction buffer [final concentration 1.0% CHAPS (CalBiochem), 0.25 mM phenylmethyl sulfonyl fluoride, muM EDTA, 0.05 M Tris (pH 0.1 M NaC1, 100 pM leupeptin] and incubated for 1 hour at 4 0 C with agitation. Commercially obtained antibodies (CalBiochem, Boehringer Mannheim) directed against human plasma proteins (20 pl) were bound to protein A-agarose beads pl, Repligen) for 1 hour in CHAPS extraction buffer on ice. The beads were washed two times with detergent wash buffer [CHAP extraction buffer plus 1% deoxycholic acid and 0.1% SDSJ and were incubated with the labeled medium overnight at 4 0 C with agitation. The beads were pelleted and washed three times with detergent wash buffer. Bound proteins were eluted with 50 pl electrophoresis sample buffer containing 2% SDS and 2% 2-mercaptoethanol, heated at 100 0 C for 10 minutes and analyzed by SDS-PAGE. Gels were processed for WO 90/10060 PCT/US90/00915 13 fluorography with Autofluor (National Diagnostics), dried, and autoradiographed at -85 0 C on XAR-5 film.

This analysis suggested that the amount of plasma proteins synthesized in vitro reflected the concentrations found in plasma. The intensities of the polypeptide bands in descending order were albumin, alpha 1 antitrypsin, fibrinogen, transferrin, apo A-I and E, beta 2 microglobulin, pre-albumin, apo A-II and A-III, complements C3, C4 and C5, C-reactive protein, and apo C-2 and C-3. All markers examined were detected with the exception of alpha fetoprotein, which is a marker for poorly differentiated fetal or malignant liver tissue.

The expression of numerous plasma proteins indicated that differentiated hepatocytes of parenchymal origin were maintained in culture.

Hepatocyte cultures grown on coverslips were analyzed at various times during the culture period for the presence of a novel NANBH virus-associated antigen that cun be detected by immunocytochemical staining (Burk et al., "Dntection of non-A, non-B hepatitis antigen by immunocytochemical staining," Proc. Natl. Acad. Sci.

U.S.A. 81:3195-3199 (1984)). Typical cytoplasmic staining was observed in all samples examined with a tendency for the percentage of cells expressing this marker to increase with time in culture. However, the number of cells with definitive staining never increased above The active replication of the virus in tissue culture was suggested by the presence of a NANBH virus-associated cytoplasmic antigen. In addition, the degeneration of the primary chimpanzee hepatocytes after 4 weeks of culture may have been due to the replication of the virus. Based on these findings, the production of infectious NANBH virus in the hepatocyte cultures was assayed by inoculation of a chimpanzee with tissue culture medium and monitoring the animal for disease manifestation. Previous experimentation with HBV suggested that the limited number of cells infected in WO 90/10060 PCr/US90/00915 14 cultures resulted in lower viral titers than those observed in vivo. In addition, it was unknown whether the expression of the NANBH agent was transient during the culture period. Therefore, media samples from each Lime point (days 3-31) were pooled and concentrated eight-fold by ultrafiltration and used to inoculate an HIBV immune, NANDH virus non-immune chimpanzee (PTTx196).

EXAMPLE 3 lissue Culture-Derived NANBH Virus Inoculum Tissue culture medium as described in Example 2 was collected at two-day intervals and passed through 0.45 pm filters and stored at -100°C. Equal amounts of each sample, days 3 through 31, were collected (190 ml total) and concentrated by pressure dialysis under N 2 gas at 4 0 C with an exclusion membrane of 30,000 MW Amicon) The eight-fold concentrate (22 ml) was stored at -100 0 C until use as exemplified by Example 4.

EXAMPLE 4 Indulction of In Vivu NANBH Virus Infection Using NANBH Hepatocyte Cell Culture Medium Without a definitive probe to monitor NANBH viral expression in the medium of these hepatocyte cultures, it was necessary to obtain conclusive evidence for the active replication of NANBH virus by the induction of hepatitis in a chimpanzee with medium derived from the virus-infected cultures.

PTTxl96, a 12-year-old male chimpanzee, received 10 ml of an eight-fold concentrate of tissue culture medium (Example 3) derived from hepatocyte cultures isolated during the acute phase of the experimental NANBH virus infection of PTTx7. A second inoculum of the same material (7 ml) was administered 12 weeks later.

Weekly blood samples and periodic liver needle punch biopsies were taken for analysis. A slight increase in ALT occurred on week 4 and microscopic examination of a liver punch biopsy at this time revealed minimal foci of WO 90/10060 P(7Y/US90/00915 15 hepatocellular necrosis with two or three neutrophils associated with the necrosis. This represents a minimal change that can be occasionally observed in normal tissue but was of interest under these conditions. An ALT/AST inversion occurred on week 8 and a second liver needle punch biopsy taken at this time exhibited essentially normal tissue with no microscopic lesions recognized.

Similar findings of normal tissue were observed in biopsy material taken on week 12.

Due to the delay in onset of clinical hepatitis, a second injection of the same inoculum (7 ml) was administered on week 12. This was followed by an elevation in ALT values beginning 3 weeks later. A persistent ALT elevation was observed 16-24 weeks after tle first inoculation. The long incubation period may reflect the low titer of our initial inoculum. However, microscopic examination of a liver punch biopsy taken on week 14 exhibited signs of hepatitis. Foci of inflammatory cell accumulation were present in the hepatic parenchyma. Occasionally there were necrotic hepatocytes (Councilman bodies) associated with the inflammation.

Kupffer cell hyperplasia was evident throughout the liver. There was also hydropic degeneration of hepatocytes in central vein areas. These changes were minimal but similar to those seen in the biopsy taken on week 4. Examination of liver punch biopsy material taken on week 17, during the period of elevated serum ALT, indicated acute hepatitis characterized by hydropic degeneration with loss of hepatocytes in centrilobular areas.

For electron microscopy, the liver biopsy was fixed with cold 3% glutaraldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.4) and postfixed for 1 hour at 4°C in 1% osmium tetroxide. Dehydration in ethanol and propylene oxide was followed by embedding in Epon 812. Sections were cut with a diamond knife on an LKB UM I ultramicrotome, stained with saturated aqueous uranyl acetate and lead citrate, and examined with an AEI EMGB electron WO 90/10060 PC/US90/00915 S16 microscope. Magnification scales were calibrated using a carbon grating replica (54,800 lines/inch); E. F. Fullam Inc., Srhenectady, Ele'tron microscopy performed on a liver biopsy taken on week 17 revealed the presence of convoluted tubules in the cytoplasm of the hepatocytes. The presence of these tubules has been used as a diagnostic marker for NA ;BH in chimpanzees. These results further substantiate that the clinical disease was due to inoculation with NANBH virus, derived from the tissue culture medium.

Plasma samples taken from PTTxl96 on weeks 0, 16 and 22 of this experimental NANBH infection were analyzed for seroconversion in response to CMV, EBV, HBV, HSV and spumavirus. These agents may cause hepatitis or be transmitted by this methodology. No increase in antibody titer was observed by specific assay for CMV, EBV, HBV, spumavirus, and HSV. These results confirm that the disease transmitted to PTTxl96 was caused by an NANBH agent.

Without a definitive probe to monitor viral expression in the medium of these hepatocyte cultures, it: was necessary to obtain conclusive evidence for the active replication of NANBH virus by the induction of hepatitis in a chimpanzee with medium derived from the infected cultures. The possibility that the infectious virus detected in the tissue culture medium was residual virus present in the hepatocytes at the time of isolation is extremely remote. Extensive washing occurred during the perfusion/collagenase procedure (2 liters) and the pelleting and resuspension (4 times) of the hepatocytes prior to plating. In addition, by day 3 in culture four changes of medium had been performed. Thus, this experiment documents the feasibility of culturing hepatocytes isolated during the acute stages of an experimental NANBH virus infection. This system should prove beneficial for identifying and characterizing the NANBH agent, leading to the elucidation of its mechanism of replication and persistence in chronic infection.

WO 90/10060 PCT/US90/00915 1.7 Those skilled in the art who have the benefit of this disclosure will recognize that changes in the formulation of the serum-free medium of the present invention can be made without compromising tle ability of the media t-, support the long-term culture of NANBH virus-infected primary hepatocytes. All such changes are considered to be within the spirit and scope of the present invention as defined by the following claims.

Claims (12)

1. Cultured primate hepatocyte cells derived from non-fetal primate liver and characterized by normal hepatocyte function for an extended culture period, as evidenced by: levels of apolipoprotein A-I and apolipoprotein E secretion in culture over a 100 day culture period which are similar to that found in primate plasma; levels of albumin secreted into culture over a 100 day culture period which is similar to that found in primate plasma; and ability to support replication of parenterally- trahsmitted-NANBH virus in culture.

2. A primate hepatocyte culture containing replicating parenterally-transmitted-NNBH virus primate hepatocyte cells infected with the NANBH virus characterized by active replication of the NANBH virus for up to 19 days following infection of the cells by the virus; and :a culture medium, which is effective to maintain 25 normal hepatocyte cells, in the absence of viral infection, in a differentiated state for up to 100 days.

3. A method of replicating parenterally- transmitted-NANBH virus by the steps of: obtaining primate hepatocyte cells infected with the NANBH virus; and culturing the cells under conditions effective to maintain normal hepatocyte cells, in the absence of viral S infection, in a differentiated state for up to 100 days.

4. A method of providing an infective parenterally-transmitted-NANBH viral composition, obtaining hepatocyte cells infected with the NANBH virus; culturing the cells under conditions effective to maintain normal hepatocyte cells, in the absence of viral infection, in a differentiated state for up to 100 days; and producing from the cultured cells said composition. The method of claim 4, wherein said culturing includes use of a serum-free tissue culture medium, and said producing includes concentrating the tissue culture medium obtained from the supernatant of the cultured cells.

6. The method of claim 4, wherein said producing includes lysing the cultured cells to obtain the NANBH composition.

7. A viral composition obtained by the method of claim 4. 25 8. A viral composition obtained by the method of claim S

9. A viral composition obtained by the method of claim 6. A cell according to claim 1 substantially hereinbefore described with reference to the examples.

11. A culture according to claim 2 substantially hereinbefore described with reference to the examples.

12. A method according to claim 3 substantially hereinbefore described with reference to the examples.

13. A method according to claim 4 substantially hereinbefore described with reference to the examples. DATED: 10 June, 1993 PHILLIPS ORMONDE FITZPATRICK Attorneys for: SOUTHWEST FOUNDATION FOR BIOMEDICAL RESEARCH I I '39 ,i 20 INTERNATIONAL SEARCH REPORT International Aoplicaiion No. PCT/US90/00915 I. CLASSIFICATION OF SUBJECT MATTER (1i several classfic.tion symools aoply, indicate all) 6 According to Internallonal Patent Clasr:fication (IPC) or to both National Classfication and IPC C12N 7/00; C12Q 1/70, 1/02; A01N 63/00 U.S. C 435/235, 5, 29; 424/93 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols U.S. CL: 435/235,5,29,240.31,948;424/93 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched a DATA BASES: CHEMICAL ABSTRACTS (CAS) 1967-1990; BIOLOGICAL ABSTRACTS (BIOSIS) 1969-1990. SEE ATTACHMENT FOR SEARCH TERMS. III. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category f Citation of Document, 11 with indication. where appropriate, of the relevant passages z2 Relevant to Claim No. Y Biological Abstracts, Volume 733, Issued 1981, BURK 1-6,8-13 et al, "Ultrastructural changes and Virus -like 20-26 particles localized in Liver Hepatocytes of CHIMPANZEES infected with Non-A Non-B Hepatitus", Abstract 18396. See entire Abstract Y The Journal of Biological Chemistry, Volume 261, No.8, 1-6,8-13 Issued 15 March 1986, Edge et al, "Cultured human 20-26 hpatocyles" pages 3800-3806. See entire Article. Y In Vitro Cellular and Developmental Biology Volume 24 1-6,8-13, No. 3, March 1988, Salas-Prato et al, "Attachment and 20-26 multiplication, Morphology and protein production of human fetal primary liver cells cultured in hormonall defined media", pages 230-238. See entire Article. Y Biochemical Journal, Volume 243, issued 1987, Diaz- 14-15 Gil er al," Identification of a liver growth factor as an albuminbilirubin complex", pages 443-448. See entire Article X US, A 4,464,474 (GOURSAGET et al) 07 AUGUST 1984 16 19 Y See entire document.

17-18 Special categories of cited documents: to later document published after the international filing date document defining the general sate of the art wiich is not or priority date and not in confict with the application but considered to be of particular relevance cited to understand the principle or theory underlying the illventfon earlier document but ublished on or alter the International document of particular relevance the claimed invention Scannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to Involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Siearch Report 04 MAY 1990 12 JUN 1990 International Searching Authority ia ure of AuthorizOfficer ISA/US 4 AIL E. POULOS Fom PCTISA/210 (econd ashik (Rev.t11-7) International Aoplication No. PCT/US90/00915 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET Y In Vitro, Volume 18, No. 1, Issued January 1982, 7-14 Marceau et al,"Growth and Functional Activities of neonatal and adult rat hepatocyles cultured on fibronectin coated substratum in serum-free medium" Pages 1-11, See entire article. Y In Vitro cellular and Developmental Biology, Volume 25 1-6,8-13. No. 2, Issued February 1989, Landford et al,

20-26 "Analysis of plc na protein and lipo protein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium" pages 174-182. See entire article. OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This international searcn report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1.1 Claim numbers .because they relate to subject matter I' not required to be searched by this Authority, namely: Claim numbers because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out Ia, specifically: 3. I Claim numbers because they are dependent claims not drafted in accordance with the second and third sentences of PCT Rule 6.4(a). VI.[ OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Slarching Authority found multiple inventions in this International applicaion as follows: 1.1 As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the international application. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the International application for which fees were paid, specifically claims: 3.1] Nc required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims: it is covered by claim numbers: 4. As all searchable claims could be searched without effort justifying an addilronal fee, the International Searching Authority did not invite payment ol any additional Ice. Remark on Protest M The additional search fees were accompanied by applicant's protest. L] No protest accompanied the payment of additional search fees. Form PCTSA,210 (suppam l shim (Rev. 11.87) PCr/US9O/00915 ATTACH=E'l to Form PCr/ISA/210 Part II FIELDS SEARCH-ED SEARCH TEF~bMS Hepatocytes Serum free Lirioleic Liver growth factor EGF Prolactin Non A nomn B hepatitis Virus J.R. Jacob R. E. tLandf ord K.H. Burk