HYBRIDOMA Volume 11, Number 4, 1992 Mary Ann Liebert, Inc., Publishers
Hybridoma Generation by Hypo-Osmolar Electrofusion:
Characterization of Human Monoclonal Antibodies to Schistosoma mansoni Parasite Antigens GERD KLOCK,2 ADAM V. WISNEWSKI,1 EMAD A. EL-BASSIOUNI,3 MOHAMED I. RAMADAN,3 PETRA GESSNER,2 URLICH ZIMMERMANN,2 and THOMAS F. KRESINA1
'Department of Medicine, Program of Geographic Medicine,
Miriam Hospital, Brown University International Health Institute, Providence, RI
2Institute of Biotechnologie, University of Wurzburg, Wurzburg, Germany
3Department of Medicine, University of Alexandria, Arab Republic of Egypt ABSTRACT Human monoclonal antibodies which bind Schistosoma mansoni
and egg antigens were identified and characterized from hybridomas generated using the hypo-osmolar electrofusion technique of somatic cell fusion. Splenocytes from S. mansoni infected individuals were mitogen-activated in vitro and subsequently fused by electrofusion. The greatest number of HAT resistant hybridomas per helical fusion chamber was obtained with unfrozen splenocytes cultured for 4-6 days after introduction of mitogen. Hybridomas secreting IgG antibodies recognizing parasite antigens were identified by ELISA. Twenty-one cloned cell lines secreting IgG antibody were maintained for at least 6 months. Characterization of antigen reactivity by Western blot analysis of nine cloned cell lines revealed antibodies which bound stage specific parasitic antigens. The data show that the technique of hypo-osmolar electrofusion produces stable, antibody producing hybridomas. The human monoclonal antibodies screened represent candidate molecules useful in the investigations of the human pathogen S. mansoni. worm
Schistosomiasis is a major health problem in developing countries with over 200 million individuals infected in developing countries (1). A major thrust of numerous laboratories has been to develop immunological and molecular biology-based approaches to control this multicellular parasite. These efforts have as their goal the development of a vaccine based on the observation of immunologically mediated resistance to infection. Murine monoclonal antibodies have been successful tools used to facilitate these research efforts. Candidate vaccine molecules (antigens) have been identified by monoclonal antibodies (2,3). These antibodies have been used to purify antigenic molecules as well as to screen cDNA expression libraries and are the basis for idiotype-based vaccine strategies (4-6). Specific murine
monoclonal antibodies can also provide passive resistance to infection in animal models of the disease. Human monoclonal antibodies have yet to be successfully utilized in the research efforts to develop an immunologicallybased vaccine in schistosomiasis. Although recent studies have reported the generation of human monoclonal antibodies in S. mansoni (7) and S. j aponicum (8) both studies utilized the Consistently polyethyene glycol somatic cell fusion technique. in our studies (8) with S. japonicum as well as in other laboratories (9,10) this technique has resulted in somatic cell fusions with poor fusion efficiencies and unstable antibody secreting hybridomas. Thus the present study details the use of hypo-osmolar electrofusion technique in the generation of human The results shown here monoclonal antibodies in S. mansoni. indicate that this technique results in high fusion ratio with the generation of stable IgG secreting hybridomas which produce molecules reactive with various parasite antigens.
MATERIALS & METHODS Human Monoclonal Antibody Formation. Human monoclonal antibodies were generated from two individuals with the chronic sequelae of Schistosomiasis. Both a 64-year old male and a 26year old female presented with bleeding oesophageal varices requiring splenectomy as part of a portal caval decompression
Preparation of Splenocytes from Human Spleen. Splenic biopsy tissue was transported at 20-25° C for 18 h, and then grinned through a 60 mesh sieve screen to create a crude splenocyte preparation and 250-300 ml of complete growth medium (CGM) were added. The CGM was prepared as follows: RPMI 1640 medium (Biochrom, FRG) was supplemented with 10% fetal calf serum (Boehringer, FRG), 2 mM L-glutamine (Biochrom, FRG), 2 mM sodium pyruvate (Biochrom, FRG) nonessential amino acids (lx, Boehringer, Mannheim, FRG), 100 units/ml pen-strep (Seromed, FRG). Storage of Crude Splenocytes in Liquid Nitrogen. Splenic
cells were sedimented at lOOxg for 20 min and washed 1-3 times in RPMI 1640 medium, containing 8 units/ml heparin, 100 units/ml of pen-strep (Seromed, FRG). Aliquots were resuspended in freezing medium (10% DMSO in FCS, cell density 4 x 167 cells/ml) and were stored frozen for 1 day at -80° C. Then, the cells were transferred into liquid nitrogen and were stored at -196° C for
up to 3 months.
The splenocytes Culture and Mitogen Stimulation. cultured for 4-8 days at 37° C in a 5% CO"1 enriched atmosphere in 24 well plates (costar), filled with 1 ml of CGM
containing 2.4 pg/ml phytohaemagglutinin M (PHA-m, Seromed, The cell density was l-3xl06 cells/ml. For mitogen stimulation, the frozen cells were thawed quickly at 37° C and were washed once in CGM containing heparin (12). Alternately purified splenocytes were directly mitogenThe splenocytes were cultured for stimulated without freezing. 4-8 days at 37° C in a 5% C02 enriched atmosphere in 24 well plates (costar) filled with 1 ml of CGM containing 2.4 ug/ml phytohaemagglutinin M (PHA-m, Seromed, Berlin, FRG). The cell density was l-3xl06 cells/ml.
Hypo-osmolar Electrofusion of Human Spleen Lymphocytes with Cells from the Mouse-Human Heteromyeloma Lines H73CII or HAB-1
lymphocytes and heteromyeloma cells H73CII (12) HAB-1 (13) were washed (lOOxg for 10 minutes) in L375-fusion medium containing 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.1% bovine serum albumin (Serva, FRG # 11930), and The sorbitol sufficient to adjust the osmolarity to 75 m osm. fusion partners were mixed in L375-fusion medium in a ratio of 2 lymphocytes per heteromyeloma cell to a final cell concentration of 5*10* lymphocytes and 2.5*106 heteromyeloma cells (H73CII or HAB-1) per ml. 200 ul of the cell mixture was filled into a standard helical fusion chamber (14). After filling, the chambers were connected to a Biojet CF electrofusion power supply (Biomed, Theres, FRG), The cells were aligned using an alternating field of 250 V/cm at a frequency of 1.5 MHz for 30 sec. Then, a single square wave fusion pulse of 1250-1750 V/cm and 15 usec duration was applied. After 10 minutes the chambers were rinsed with 1 ml CGM without phenol red and the cells were plated into 4 wells of a 24-well plate prefilled with CGM (without phenol red) (Greiner). The plates were then cultured at 37° C in a 5% C02 enriched atmosphere. After 24 h 1 ml of CGM supplemented with HAT (Boehringer, FRG) were added to each well. After 8-10 days in culture, discrete hybridoma clones could be detected microscopically. Approximately 3 weeks after fusion, single hybridoma clones were transferred into one well of a 24 well plate (Costar). After 8 days, 2 ml of the culture supernatants were collected and stored frozen for further PHA-m stimulated
Identification of hybridoma proteins binding parasite antigen was initially performed by ELISA (8). Parasite antigens prepared as previously described (15) were used to coat 96-well Wells were coated with lOOul of 1 mg/ml of microtiter plates.
Plates were incubated 12-24 SWAP or SEA diluted in PBS (pH7.4). hours at 4° C and blocked with 5% BSA in PBS for 2 hours at 37° C. Next lOOul of crude culture media was added and the plates were incubated for 2 hours at a 37° C in 5% C02 incubator. Following several washes, 100 ul of horseradish peroxidase (HRP)conjugated goat anti-human IgG (Chemicon), diluted 1:3000 was added for 40 min at room temperature. After extensive washing, plates were developed with a 0.1% ortho-phenyl-diamine solution in 0.1M Citrate buffer (pH4.5), and absorbance at 492nm was measured. Samples identified as positive for reactivity to parasite antigen were sub-cloned and retested by ELISA and further characterized by Western blot analysis.
Western Blot Analysis Parasite antigens were added to SDS-PAGE reducing buffer and placed in a 100° C water bath for 5 min. before electrophoresis (6) using a 10% acrylamide gel (100°C). After 4 hours electrophoresis at 25MA per gel, proteins were transblotted onto nitrocellulose as previously described (6) for 2 hours at 0.6A. Nitrocellulose strips were rocked overnight at 4 C in 5% dry milk in tris buffered saline (TBS pH 7.4). Three mis of crude hybridoma culture supernatant was added for 3 hrs. at 37 C in a 5% CO2 incubator. Strips were washed 3 times for 10 minutes with a TBS 0.1% milk buffer. Three mis of a 1:500 dilution of HRP-GT anti-Human IgG was added for 1.5 hrs. After four 10 minutes washes in wash buffer and two 5 minute washes in TBS, the strips were developed wl .15% 4-chloro-l-naphthol/.2% methanol/.15% H202 solution. -
Optimum Electric Field Parameters for Fusion of Human Splenocytes with Heteromyeloma Cells The electrical field conditions for optimum hybridization of human splenocytes with heteromyeloma cells are consistent with previously published optimal fusion efficiencies using the helical chamber and the hypo-osmolar electrofusion protocol (12,14). Differences in the fusion efficiencies achieved using fusion field strengths of 1.25 or 1.75 kVcm did not reach statistical significance for either heteromyeloma partner (results not shown). Fusion Yield For hybridization
experiments, either freshly prepared splenocytes which had been stored in liquid nitrogen were stimulated with PHA-m for up to 8 days. After hypo-osmolar electrofusion, the hybrid yield from "fresh" splenocytes was 14 HAT resistant clones per helical chamber (heteromyeloma partner H73CII). Splenocytes, which had been stored in liquid nitrogen yielded 10 clones per helical chamber (heteromyeloma partner H73CII). The respective values for hybrid yield after fusion of human splenocytes with HAB-1 heteromyeloma cells are 8 clones per helical chamber ("fresh" splenocytes) and 12 clones per chamber ("frozen" splenocytes). The hybridoma yields after electrofusion of heteromyeloma cells to human splenocytes depended strongly on the preceding mitogen activation of the splenocytes (Fig. 1). The hybrid yield of unstimulated splenocytes
o Q. u>
97,75,68 62,59,47 45,38 97,75,68, 62,59,47 45,38 97,75,68 62,59,47 45,38 97,75,68, 62,59,47 45,38
78,43,38, 26,24 97,75,70 68,47,43, 26,24
50,43, 37 26,24 75,26, 24
Parasite antigens extracted as described in the text. Western blot of antigens for all stages of parasite performed as described in the text. Data presented as molecular weight estimate of proteins reactive with specific clone on Western blot analysis. Standards used for molecular weight estimation were: Adolase 110,000, Serum Albumin 66 kDa, Trypsin Inhibitor-27 kDa, 14 kDa. Myoglobulin -
Figure 2. Western Blot Hybridization of Human Monoclonal Antibodies. S. mansoni antigens are derived from worms (W), schistosomula (S), cercariae (C) and egg (E). Pooled human serum from chroniclly infected individuals are shown (Lanes 1-4) and lOx culture supernatant from subcone SM38E10 (Lanes 5,6,7,8) are shown. The binding pattern displays the stage specificity of
analysis. The molecular weight and parasite stage reactivity of the antigens recognized by these antibodies are represented. These nine monoclonal antibodies collectively recognized multiple antigens of molecular weight ranging from 24,000 daltons to >110,000 daltons. Two patterns of antigen reactivity are readily Monoclonal antibodies, such as SM 26/6 and 38E10 bound single antigens present only in a specific stage of the parasites life cycle (Fig. 2). Alternatively, antibodies such as 37C4 and 24E8, bound to multiple parasite antigens expressed in all stages of the parasite's life cycle tested (Fig. 3). This polyreactivity apparent to multiple antigens was apparent in six of the nine Since these reagents are monoclonal antibodies tested to date. clonal it's likely that these latter monoclonal antibodies recognize a recurrent epitope(s) which modulates expression through the parasite life cycle.
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Figure 3. Western Blot Hybridization of Human Monoclonal Antibodies. Using parasite antigens derived from S. mansoni Lanes 1worms (W), schistosomula (S), cercariae (C) or egg (E). 4 are immunoblotted with pooled human serum from chronically infected individuals. Lanes 5-8 are immunobltoted with 10X concentrated culture supernatant containing hybridoma subclone SM23/11-10. Lanes 9-12 immunoblotted with lOx concentrated culture supernatant containing subclone SM23/11-24. Figure shows the virtually identical binding patterns for the two subclones and modualtion of epitope expression through the life cycle. DISCUSSION The present study is the initial description of the use of hypo-osmotic electrofusion for the generation of human IgG monoclonal antibodies binding parasite antigens of S. mansoni. In this study as well as previous studies (16,17) the protocol used results in a very high and reproducible yield of hybridomas. The present study advances the findings of previous studies by using splenocytes from individuals with chronic S. mansoni pathology and deriving human monoclonal antibodies germane to the parasite. The present study also clearly demonstrates the importance, in this biological system, for the appropriate heteromyeloma fusion pattern. All hybridomas generated to the S_^
No antigen mansoni antigens were derived from the H73CII cells. reactive clones were generated using the HAB-1 heteromyeloma cell line. However, the HAB-1 cell has been successfully used with the electrofusion technique to generate antigen reactive hybridomas in other antigenic systems (13 and unpublished
a high fusion efficiency was also found to be appropriate mitogen stimulation of splenocytes used in the fusion. Mitogen induced expression of splenocyte cell surface receptors which are complementary to cell surface receptors expressed on the hybridoma fusion partner is thus critical for efficient fusion. Under the hypo-osmotic conditions used in the fusion, the osmotic stress and associated tension created in the membrane plane of swollen cells changes their membrane structure as evidenced by changes in membrane permeability (18,19). During this time there is a high mobility of membrane components and the postulation of protein-free lipid domains. Thus cell surface receptor alignment occurs (12). High efficiency fusion with antigen reactivity would occur with membrane receptor "matching" or "complementary" between heteromyeloma and lymphoid cells. This likely occurs in the present protocol between days 4-6 after stimulation of splenocytes in vitro. These cells undergo mitogen induced differentiation in vitro at this time with augmented surface receptor expression. The present study describes mitogen-stimulation of lymphocytes. This technique potentially allows for a greater pool of activated lymphocytes for fusion rather than other techniques such as EBV activation and/or transformation. In this regard, EBV can activate B cells bearing any cell surface heavy chain (20). But EBV transformation has been reported to be restricted to immature B cells that express the EBV receptor (21). Under these conditions EBV transformation would result in a high number of IgM antibody secreting hybridomas. Previous studies (22) have shown that IgM human monoclonal antibodies are frequently low affinity polyreactive antibodies. In addition, for the case of S. mansoni, the splenocytes derived from S.mansoni patients with chronic schistosomiasis are not likely to be comprised of large populations of antigen specific EBV
transformable immature B cells.
This is due to chronic
parasitism of the host. Individuals with chronic schistosomiasis can be exposed to parasite antigens for as long as 20-40 years. Thus, the splenocytes derived from patients have been bathed in worm antigen for years and are likely differentiated to either mature B cells or antigenically tolerant B cells. Antigenically tolerant B cells would, obviously be poor producers of antibody. Mature B cells derived from individuals exposed to worm antigens over an extended period of infection would produce predominantly
monoclonal antibodies to immunodominate antigens. This later observation is precisely the data presented in this study. The data of the present study therefore suggest that the patient selection is important in the generation of human monoclonal antibodies that would be good candidates for passive protection. Such monoclonal antibodies would be directed to the cercarial and schistosomula stage of the parasite. Such murine monoclonal antibodies have been generated by others (23-25) and in our laboratory by priming animals with freeze/thawed schistosomula (26). It is unlikely that such methodologies can be used to generate human monoclonal antibodies. However, peripheral blood lymphocytes derived from acutely infected individuals with a primary infection represent a likely source of
lymphocytes exposed to cercarial and schistosomula antigens. lymphocytes would be limited in number and thus the technique of electrofusion using small numbers of lymphocytes with in vitro antigen priming represent the technique of choice. Finally, the monoclonal antibodies described in the present study are of potential value in the study of the immunoregulation of S. mansoni induced pathology or in vaccine development. Recent studies (8,27) have shown that human monoclonal antibodies generated by EBV transformation can modulate in vitro T cell Thus, these and the reagents responses to schistosome antigens. of the present study represent candidate molecules for an antipathology (granulomatous inflammation) vaccine in schistosomiasis. With regard to vaccine development, numerous studies (2,3,23-25,28-32) have defined antigens derived from S. mansoni life cycle stages. These antigens have been shown to induce resistance to reinfection (in the range of 30-70%) in permissive hosts. The reported molecular weight of some of these antigens are identical to antigens identified by Western Blot hybridization of the human monoclonal antibodies presently described. Thus, monoclonal antibody SM24E8 which recognizes a 28 kd antigen in worms potentially could cross react with an epitope on glutathione S-transferase (24 kd). Antibodies SM23/II-6-10,21,24 recognize 68,38,45 and 97/kd (paramycin) antigens all of which have been reported to induced resistance to reinfection. Further epitope mapping studies are required to B
determine if these human monoclonal antibodies bind relevant
protective epitopes on the identified antigens. With regard to vaccine therapy human monoclonal antibodies have been used to
reverse renal allograft rejection and to treat cancer, multiple sclerosis and most recently patients with rheumatoid arthritis (33-36). However, this therapy has been complicated by the development of a host immune response to the xenogenic antibody. The generation of clinically relevant human monoclonal antibodies may be affective in reducing this pathogenic response to therapy.
ACKNOWLEDGMENTS The authors are very grateful to Ms. B. Herrmann for her skillful technical assistance and to Dr. R. Schnettler for his helpful suggestions and the purification of the splenocytes. The work was supported in part by grants of the Federal Ministry for Technology and Research (BMFT grant No. 0319209 AO) and the Deutsche Forschungsgemeinschaft (SFB 176 project B%) to U.Z. and NIH grant (USPHS) AI 25167.
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Correspondence : Thomas F. Kresina, Ph.D. Department of Medicine Program of Geographic Medicine Miriam Hospital Providence, RI 02906 (401) 331-8500 X 4533
Received for publication: 1/27/92 Accepted after revision: 4/24/92