Echinococcus multilocularis: In vitro secretion of antigen by hybridomas from metacestode germinal cells and murine tumor cells

EXPERIMENTAL

PARASITOLOGY

Echinococcus Hybridomas

68,

186-191 (1989)

multilocularis: In Vitro Secretion of Antigen by from Metacestode Germinal Cells and Murine Tumor Cells

ANGELADIECKMANN-SCHUPPERT

*J ANDREAS RUPPEL~,REINHARD W)ERNERFFCANK*

BURGERS**, AND

*Department of Parasitology, University of Hohenheim, Stuttgart, Federal Republic of Germany: and fInstitute for Tropical Hygiene and Slnstitute for Immunology, University of Heidelberg, Federal Republic of Germany DIECKMANN-SCHUPPERT, A.,RUPPEL, A., BURGER, R., AND FRANK, W. 1989. Echinococcus multilocularis: In vitro secretion of antigen by hybridomas from metacestode germinal cells and murine tumor cells. Experimental Parasitology 68, 186-191. Hybrid cells were produced from Echinococcus multilocularis metacestode germinal cells and murine tumor cells. Small colonies were formed which, while ceasing to grow after a few generations, remained viable for at least 10 weeks. These hybridoma cells secrete antigen(s) reacting in indirect immunofluorescence and ELISA specifically with sera from patients suffering from an E. multilocularis infection. The antigen(s) appear suitable for the differential diagnosis of E. multilocularis and E. granulosus. Thus, hybridoma cells may produce helminth antigens. 0 1989 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Echinococcus multilocularis; Echinococcus granulosus; Cestodes, Differential diagnosis, Antigens, Myeloma cells, Cell fusion; Immunofluorescence assay (IFA); Enzyme-linked immunosorbent assay (ELISA).

Human alveolar echinococcosis is a usually fatal disease resulting from the development of metacestodes of Echinococcus multilocularis. Diagnosis is normally performed by a variety of serological tests detecting antibodies against this parasite in the patients’ blood. Since the antigens for such tests are commonly derived from E. granulosus cysts obtained from infected cattle, assays performed with antigens from such variable sources are difficult to standardize. Moreover, the serological crossreactivity between the two Echinococcus species does not allow a differential diagno’ Present address and to whom correspondence should be addressed at Department of Structural Biology, Biocenter, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. * Present address: Bundesgesundheitsamt, RobertKoch-Institut, Berlin, FRG.

sis using the presently available serological assays. A species-specific test would be particularly relevant to improve both prognosis of the disease and therapy schedules. Standardization and differential diagnosis appear to become feasible using a defined antigen (Em2a) affinity purified from E. muftilocularis metacestodes (Gottstein et al. 1983; Gottstein 1986). Alternatively, a species-specitic antigen of constant quality might be produced by in vitro culture methods. Whereas differentiation of Echinococcus protoscoleces to adult worms can be achieved in vitro @myth 1962; Smyth et al. 1966), larval Echinococcus material could be maintained, but the cells failed to grow continuously in culture (Lukashenko 1964; Sakamoto 1978; Rubino et al. 1983). The primary culture in vitro of cells derived from another helminth, Schistosoma mansoni, has already led to the formation of small colonies (Weller and Wheeldon 1982). 186

0014-4894/89 $3.00 Copyright 0 1989 by Academic Press, Inc. Au rights of reproduction in any form reserved.

Echinococcus:

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TUMOR HKWDOMAS

Since growth of E. multilocularis metacestode germinal cells in primary culture was poor (Dieckmann and Frank 1988b), we have attempted to induce continuous growth of these cells in vitro by fusing them to a murine tumor cell line. MATERIALS AND METHODS Cells. The Echinococcus multilocularis strain was originally established by oral infection of common voles (Microtus arvalis) with eggs from the gut of a naturally infected fox (Vulpes vulpes) (Bosch 1982). Metacestode tissues were subsequently passaged in jirds (Meriones unguiculatas) by intraperitoneal injection (Hinz 1972). Single cell suspensions of E. multilocularis germinal cells were obtained from infected jirds according to a previously described procedure (Dieckmann and Frank 1988a). Contaminating erythrocytes were lysed with isotonic ammonium chloride. The P3-X63-Ag8.653 mouse myeloma cell line (Kearney et al. 1979) was maintained at 37 C, 100% relative humidity, and 7% CO, in DMEM (Flow Laboratories, No. 12-322-54, Irvine, Scotland, UK) supplemented with 10% heat-inactivated fetal calf serum (Seromed, No. SO115, Biochrom KG, Berlin, FRG), glutamine, and penicillin/streptomycin according to Burger et al. (1982). Viability of cells was tested by exclusion of Trypan blue and by uptake and cleavage of 0.25 &ml fluorescein diacetate. Fusions. These were performed according to the method of Oi and Herzenberg (1980) as modified by Burger et al. (1982). Briefly, 3 x lo7 E. multilocularis germinal cells and the same number of tumor cells were pelleted together and 1 ml of 50% polyethylene glycol4000 (Merck, Darmstadt, FRG) in DMEM was added dropwise over a period of 60 sec. After fusion, the cells were. cultured in flat-bottom microtiter plates (8 x 10” cells/well) in selective medium (HAT, i.e., DMEM maintenance medium as described above, supplemented additionally with hypoxanthine, aminopterine, and thymidine according to Burger et al. (1982). In 10 independent experiments a total of 35 X 10’ E. multilocularis germinal cells were processed. ZFA. Cells were probed by IFA for the expression of E. multilocularis antigen on their surface. They were incubated with alveolar echinococcosis patient serum diluted 1:108 in HAT medium. After 30 min at 37 C, cells were washed twice and incubated under the same conditions with FITC-labeled sheep anti-human IgG antibodies (Wellcome Research Laboratories, Beckenham, UK). Cytoplasmic antigens were evaluated according to the same protocol after cytocentrifugation and acetone fnation of the cells. Control experiments were performed with individual sera from patients suffering from various parasitic or other diseases and with a pool of 50 normal human sera instead of alve-

olar echinococcosis patients’ sera. Expression of murine surface immunoglobulm by the hybridomas was tested by direct incubation with FITC anti-mouse IgG antibodies (Wellcome). ELZSA. Polystyrene microtiter plates were coated with 2 pg protein/ml of sonicated hybridoma cells in 10 r&f carbonate buffer, pH 9.6. Coating was performed overnight at room temperature. The plates were washed three times with saline and used immediately. An Enzygnost kit (Behring, Marburg, FRG) was used for the assays. Subsequent incubations with test serum, galactosidase-labeled anti-human antibodies and enzyme substrate, as well as the spectrophotometric evaluation of the test, were performed according to the instructions of the manufacturer. PAS staining. Cytocentrifuged preparations of the hybridomas were fixed with 70% ethanol and stained for glycoproteins by the periodic acid-Schiff stain (Romeis 1968). RESULTS

Hybridoma cells were maintained in culture and remained viable for at least 10 weeks, at which time the experiments were terminated. Two weeks after the fusion, numerous small colonies were formed (Fig. 1). Within the next 4 weeks, these colonies reached a size of 60-300 cells. At later times cells ceased to divide but remained viable until the end of the experiments. In control experiments either Echinococcus multilocularis or myeloma cells were fused to themselves. No colonies formed during subsequent culture and the cells died within 14-20 days. Neither did colonies appear when the parental cells were cultured in

FIG. 1. Colony of hybrid cells 2 weeks after the fusion of Echinococcus multilocularis germinal cells with murine myeloma cells. (Bar = 50 pm.)

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HAT medium without having previously been subjected to fusion. Thus, colony formation required the presence of both E. multiloculuris and tumor cells in the fusion. The hybrid cells were surrounded by secreted material giving a positive PAS reaction. This material reacted strongly in the IFA with sera from patients with alveolar echinococcosis (Fig. 2a). In addition, the surface of the cells themselves showed a few fluorescent spots. These were also observed upon performance of the test in the presence of azide (0.01%) or at 0 C, suggesting that capping of antigen was not induced by the IFA procedure. With normal human serum, however, fluorescence was neither detectable on cell secretions nor on cell surfaces (Fig. 2b). Sera from patients with cystic echinococcosis (E. granulosus infections) yielded only a very weak fluorescence on the secretions (Fig. 2~). This heterologous reaction with the secreted E. multilocularis antigen was clearly distinguishable from the strong reaction seen with sera from patients with the homologous E. multilocularis infection. E. multilocularis, but not E. granulosus patient sera, also reacted with cytoplasmic antigens of the hybridoma cells (Figs. 3a,b, respectively). These observations suggest that the antigens were synthesized by the hybridoma cells rather than introduced into the cultures by the parental E. multilocuhis cells, Sera from patients suffering from a variety of other diseases failed to react in the IFA. The ELBA readings essentially confirmed the IFA results (Table I). The presence of murine antigens on the hybridoma cells was evaluated by direct treatment with FITC anti-mouse IgG conjugate. A specific, albeit faint, fluorescence was observed with the hybridomas as well as with the parental myeloma cells, but not with E. multiloculuris germinal cells. No reaction was seen with an anti-human IgG conjugate, which was tested as a negative control.

ET AL.

FIG. 2. Indirect immunofluorescence assay for Echinococcus antigen performed with viable hybrid cell colonies. Assays were done with (a) serum from a patient infected with E. multilocularis, (b) a pool of sera from 50 healthy blood donors, and (c) with serum from a patient infected with E. granulosus. (Bars = 50 w.)

Parasite antigens were detectable on the surface of 87 f 6% of the colony cells, while murine antigens were observed on 65 + 8% (eight determinations each). Thus, si-

Echinococcus:

TUMOR

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HYBRIDOMAS

TABLE I Reactivity of Human Sera with the Hybridoma-Derived Antigen in Indirect Immunofluorescence Assay and ELISA Intensity of reaction

Disease of patients Echinococcus multilocularis E. granulosus (10) Taenia sp. (intestinal) (4) T. solium (cysticercosis) (2) Spirometra sp. (3) Ancylostoma duodenale (4) Ascaris lumbricoides (5) Loa loa (3) Onchocerca volvulus (2) Strongyloides stercoralis (2) Toxocara canis (3) Trichuris trichiura (5) Fasciola hepatica (2) Schistosoma mansoni (3) Entamoeba histolytica (3) Toxoplasma gondii (3)

Liver cirrhosis (2) Rheumatic arthritis (2) Elevated IgE (3) None (pool of 50 sera)

FIG. 3. Indirect immunofluorescence

assay for cyantigen performed with actoplasmatic Echinococcus etone-futed hybrid cells using serum (a) from a patient infected with E. multilocularis and (b) from a patient infected with E. granulosus. (Bars = 10 pm.)

(10)

+++ + -

-

D Number of sera tested.

secretory antigens from protoscoleces maintained in vitro as described by Auer and Aspock (1985). Although the limited amount of material enabled us to perform only two experiments, the results (not illustrated) suggestthe presence of proteic components in the secreted antigens. The potential for in vivo growth of the hybridomas was tested in five nude Balb/c mice which each received lo6 cells subcutaneously and were killed 3 months later. Several small nodules developed under the skin of one animal, but these did not express Echinococcus antigen asjudged by an immunofluorescence test with alveolar echinococcosis patient serum.

multaneous expression of both antigens occurred in at least half of the hybridoma cells. In order to further characterize the secreted antigen(s), colonies were harvested, washed free of serum, and subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. Reactions with E. multilocularis patient serum were obtained at positions corresponding to molecular weights of about 1829, and 42 kDa. No reaction was seen with an antiserum raised in Meriones unguiculatus against the defined Em2 antigen (kindly provided by Dr. B. Gottstein, Zurich). The bands obDISCUSSION tained with the E. multilocularis patient serum had positions in the blots differThis report describes the production in ent from those produced by excretory- vitro of helminth antigen(s) by hybridomas

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from murine

tumor cells fused to Echinocells. This experimental approach differs from the few previous attempts at expressing parasite antigens in hybrid cells. Hybrids between Fusciolu hepaticu cells and rat fibroblasts remained viable in culture, but failed to express detectable parasite antigen (Howell 1981). More recently, expression of F. heputicu antigens on Cl27 mouse cells was achieved upon their transformation with purified F. heputicu DNA fragments (Beardsell and Howell 1987). When the protozoan parasite Trypunosomu cruzi was fused in vitro to bovine embryo skeletal muscle cells, parasite antigens were expressed on the cell surface (Crane and Dvorak 1980). The production of parasite antigens in our system appears to be more substantial than in either one of the abovementioned studies. In fact, most of the Echinococcus antigen was detected as extracellular deposits, whereas membraneassociated antigen was relatively scarce. Since the colonies of the hybridoma cells remained small, only a preliminary characterization of the secreted antigens was possible. However, the data do not suggest an identity of the hybridoma-derived antigen with either one of the previously described species-specific E. multiloculuris antigens (Auer and Aspock 1985; Gottstein et al. 1983). A chromosome analysis of the hybridomas was not attempted. We have no continuously growing cell line of E. multiloculuris germinal cells available (Dieckmann and Frank 1988b) which might be used to establish the karyotype of this species. Also, germinal cells from Echinococcus grunulosus were recently reported to have numbers of chromosomes varying from 40 to 90, although protoscoleces had 2n = 18 (Fiori et al. 1988). Since loss of murine chromosomes should be expected in our germinal cell-derived hybrids, we anticipate considerable difficulties in establishing their karyotype.

ET AL. ACKNOWLEDGMENTS

coccus multiloculuris germinal

This study received financial support through the Deutsche Forschungsgemeinschaft (Fr 212/30-l) and the Forschungsschwerpunkt HD 19. A.D. was a Ph.D. scholar of the Studienstiftung des deutschen Volkes. Dr. B. Gottstein, Institute for Parasitology, Zurich, kindly provided some Meriones-anti Em2 serum. We thank Mrs. A. Bader and Mrs. A. Rilskens for expert technical help with some experiments. REFERENCES AUER, H., AND ASP&K, H. 1985. Studies on antigens from in vitro cultivated protoscolices of Echinococcus multilocularis and their possible use in the serodiagnosis of human echinococosis. In “Proceedings of the 2nd International Symposium on Taeniasis/Echinococcosis” (J. Prokopic, Ed.), pp. 7-15. Ceske Budejovice/Cekoslovakia. BEARDSELL, P. L., AND HOWELL, M. J. 1987. Production of parasite antigen by co-transformation. Parasitology Today 3, 28-29. BOSCH, D. 1982. Tierexperimentelle Untersuchungen zur Entwicklung von Echinococcus multilocularis. In “Probleme der Echinokokkose unter Berticksichtigung parasitologischer und klinischer Aspekte” (R. Blhr, Ed.) Huber, Bern. Aktuelle Probleme in Chirurgie und Orthopiidie 23, 36-40. BURGER, R., DEUBEL, U., HADDING, U., AND BITTER-SUERMANN, D. 1982. Identification of the functionally relevant determinants on the complement component C3 with monoclonal antibodies. Journal of Immunology 129, 2042-2050. CRANE, M. S. J. AND DVORAK, J. A. 1980. Vertebrate cells express protozoan antigen after hybridization. Science 209, 194-196. DIECKMANN, A. AND FRANK, W. 1988a. Isolation of viable germinal cells from Echinococcus multilocularis. Parasitology Research 74, 297-298. DIECKMANN, A. AND FRANK, W. 1988b. Versuche zur in vitro Kultivierung von Echinococcus multilocularis Keimschichtzellen. Mitteilungen der Gsterreichischen Gesellschaft fiir Tropenmedizin und Parasitologie 10, l-8. FIORI, P. L., MONACO, G., SCAPPATICCI, S., PUGLIESE, A., CANU, N., AND CAPPUCCINELLI, P. 1988. Establishment of cell cultures from hydatid cysts of Echinococcus granulosus. International Journal for Parasitology 18, 297-305. GOTTSTEIN, B. 1986. Purification and characterization of a specific. antigen from Echinococcus multilocularis. Parasite Immunology 7, 201-212. GOTTSTEIN, B., ECKERT, J., AND FEY, H. 1983. Serological differentiation between E. multilocularis and E. granulosus infections in man. Zeitschrift fur Parasitenkunde 69, 347-356.

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HINZ, E. 1972. Die Aufbereitung des Infektionsmaterials fur die intraperitoneale Infektion der Maus mit Echinococcus multilocularis. Tropenmedizin und Parasitologie 23, 387-390. HOWELL, M. J. 1981. An approach to the production of helminth antigens in vitro: The formation of hybrids between Fasciola hepatica and a rat tibroblast cell line. International Journal for Parasitology 11, 235-242. KEARNEY, J. F., RADBRUCH, A., LIESEGANG, B., AND RAJEWSKI, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. Journal of Immunology 123, 15481550. LUKASHENKO, N. P. 1964. Study on the development of Alveococcus multilocularis (Leuckart, 1863) in vitro (in Russian). Meditsinskaya Parazitologiya y Parazitarnye Bolezny 33, 271-278. Or, V. T., AND HERZENBERG, L. A. 1980. Immunoglobulin producing hybrid cell lines. In “Selected Methods in Cellular Immunology” (B. Mishell and S. Shiigi, Eds.), pp. 351-372. Freeman, San Francisco. ROMEIS, R. 1968. “Mikroskopische Technik,” pp. 275-276. Oldenbourg, Munich/Vienna.

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RUBINO, S., FIORI, P. L., AND LUBINU, G. 1983. The cytoskeleton of hydatid cyst cultured cells and its sensitivity to inhibitors. European Journal of Cell Biology 30, 182-190. SAKAMOTO, T. 1978. Development of echinococcal tissue cultured in vitro and in vivo. Memoirs of the Faculty of Agriculture of the Kagoshima University 14, 109-113. SMYTH, J. D. 1%2. Studies on tapeworm physiology. X. Axenic cultivation of the hydatid organism, E. granulosus; establishment of a basic technique. Parasitology 52, 441-457. SMYTH, J. D., HOWKINS, A. B., AND BARTON, M. 1966. Factors controlling the differentiation of the hydatid organism, Echinococcus granulosus, into cystic or strobilar stages in vitro. Nature (London) 211, 1374-1377. WELLER, T. H., AND WHEELDON, S. K. 1982. The cultivation of cells derived fromadtdt Schistoxntna mansoni. I. Methodology; criteria for evaluation of cultures; and development of media. American Journalfor Tropical Medicine and Hygiene 31,335348. Received 16 May 1988; accepted with revision 5 October 1988