Parasite Immunology 1992,14,451-456
Brief communication
Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization MARKUS KAMBER*f, MICHAEL BLACKMAN§, PECK-SUN LIN*, JAMES BROWN?, HILTON WHITTLE7 & RUPERT SCHMIDT-ULLRICH' *Department of Radiation Oncology, Medical College of Virginia, Richmond, VA, USA and t Medical Research Council Laboratories. Fajara, near Banjul, The Gambia Accepted for publication 29 January 1992 Summary Nine human monoclonal antibodies (MoAbs) recognizing 7 different antigenic structures of blood-stages of the human malarial parasite P.fakiparum (Pf) were produced by Epstein-Barr virus transformed B-cell lines (EBV-TCL) with or without fusion to the lymphoblastoid cell line KR4. The peripheral blood B-lymphocytes were obtained from 8 Gambian donors immune to Pf malaria. Two of the EBV-TCL could be expanded and maintained for more than 6months but neither one could be cloned. Six additional EBV-TCL were stabilized after fusion with the KR4 lymphoblastoid cell line. All resulting hybridomas permitted easy cloning. Some of the MoAbs produced distinct fluorescent staining patterns of asexual Pf blood-stage parasites when using high-resolution digitized videointensified fluorescence microscopy. Antigens on 195 kD and 155 kD proteins were. recognized by 3 and 1 MoAb, respectively, using Western blotting and immunoprecipitation techniques.
Keywords: Plasmodium falciparum, hybridoma; human monoclonal antibodies
The immune response to the human malarial parasite Plasmodium fakiparum (Pf) is mediated by both humoral and cellular reactions. Therefore, the identification and characterization of immunogenicPf blood-stage antigens (Ag) is one essential step before a protective immune response can be studied at the molecular level. Ideal reagents for the immunochemical characterization of Pf Ag would be human monoclonal antibody (MoAb) produced by B-lymphocytes from the immune human host. Several studies showed that MoAb to Pf antigens could be generated but that the yield of Ab producing Correspondence: Rupert Schmidt-Ullrich. $ Present address: Hoffmann-La Roche & Co., CH-4002 Basel, Switzerland. 8 Present address: National Institute for Medical Research, Division of Parasitology, The Ridgeway, Mill Hill, London NW7 IAA, UK.
45 1
452
M.Kamber et al.
cells was low and their stability is secreting Ab was limited (Fine et al. 1987, Lundgren et al. 1983, Schmidt-Ullrichet al. 1986, Udomsangpetch et al. 1986). The present study was undertaken in an attempt to define conditions for better sources of Pf-specific human MoAb. In comparingEpstein-Barr virus (EBV) transformed B-cells from donors immune to Pf and hybridomas derived from transformed B e l l s fused with the lymphoblastoid cell line KR4 (LCL; Kozbor et al. 1982) we examined their relative value as sources for high-yield and stable MoAb production. The human MoAbs produced were characterized in relation to Ag recognition by indirect immunofluorescence, immunoblotting, immunoprecipitation, and their activity in inhibiting Pf growth in vitro. Peripheral blood mononuclear cells (PBMC) from 8 Gambians donors, immune to Pf malaria with no recent episodes of acute malaria, were successfullytransformed with EBV (supernatant of mycoplasma-free 895-8 cells, 10 transforming units/cell; Miller & Lipman 1972) and subsequentlycultured according to a method described by Brown et al. 1986. These EBV-transformed cell lines (EBV-TCL) producing Ab against Pf antigens were subcultured by micromanipulation of individual cell clumps and finally expanded into T-25 tissue culture flasks. Mononuclear cells isolated from splenic tissue using FicollPaque gradients (Pharmacia LKB, Uppsala, Sweden), were obtained from an adult Gambian undergoing splenectomy and were transformed by EBV (SPL3B6). EBV-TCL from 7 donors were fused with the ouabain-resistant, HAT-sensitive LCL KR4 (Kozbor et al. 1982, obtained from Dr D. Kozbor) according to a previously reported protocol (Schmidt-Ullrich et al. 1986) with the addition of 10 p~ ouabain to the HAT selection medium. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (HyClone; Logan, UT), 4 mM glutamine, 1 mM Na-pyruvate, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.4 mM non-essential amino acids. Hybridomas were grown in the same medium supplemented with 0.0025 IU/ml bovine insulin and 2.5 p~ oxaloacetic acid. Cloning of EBV-TCL and hybridomas was performed by limited dilution at 0.3 cellslwell in flat-bottom microtitre plates with or without 2 x 105/mlhuman PBMC feeder cells irradiated to doses of 30 Gy. Cloning efficiency was defined as by Waldmann et al. (1987). Clonality of the cell lines was maintained through monthly recloning at the same stringency of 0.3 cellsfwell. The amount of secreted MoAb in culture supernatants was determined by ELISA using goat anti-human immunoglobulin (Ig) pre-coated on the bottom of 96-well plates and alkaline phosphatase-derivatized class- and IgG isotype-specific Ig (Shoenfeld et al. 1982).Of more than 50 EBV-TCLs produced from 8 donors (data not shown), only 8 cell lines from 7 donors were found to secrete Pf-specific Ab and could be expanded into T75 flasks to produce sufficient cell numbers for fusion with KR4 cells. With the exception of SS2F4, all EBV-TCLscould be stabilized after fusion with the KR4 cell line. Cloning was successful for all hybridomas but only for 4 of 8 EBV-TCL. However, clonability did not necessarily correlate with the stability of cell lines (Table I). With respect to MoAb production, the EBV-TCL invariably produced higher levels of Ab on the per cell basis than the corresponding hybridomas (Table 1). Considering the much improved growth properties of hybridomas the lower level of Ab production was easily compensated for by large scale cultures. Pf-specific Ab was characterized by indirect immune fluorescence using imaging technology (Mikkelsen et al. 1988), immunoblotting (Towbin et al. 1979), immunoprecipitation (Schmidt-Ullrichet al. 1986), or growth inhibition assay (Brown et al. 1986). All
Human MoAbs against P.falciparum
Table 1. Comparisons between EBV-transformed B-cell lines and their hybridomas on growth stability and antibody production
Cell line SS2F4 E*
Hi SS2D2 E H X-509 E H AJ-D2 ES H SPL3B6 E H HJ6D6 E H FS-G2 E H FS-E7 E
H
Stability (months)
Cloning efficiency (range %)
Ab production$ (pg/ml, range)
> 10 >8
n.c. 12-33
1 0-05-0.5
8
n.c. 2.2
1*3 0.15
8
47
6.3
4.8-33
0.5-1.9
>6
n.a. 13-20
n.a. n.d.
2.6 3-22
n.d. n.d.
>5
71 11-25
1.4 0.12
5 n.e.
2-1 n.a.
n.d. n.a.
79
n.e.
n.c. n.a.
n.d. n.a.
2 >6
n.c. 6-33
n.d. n.d.
4
Brief communication
Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization MARKUS KAMBER*f, MICHAEL BLACKMAN§, PECK-SUN LIN*, JAMES BROWN?, HILTON WHITTLE7 & RUPERT SCHMIDT-ULLRICH' *Department of Radiation Oncology, Medical College of Virginia, Richmond, VA, USA and t Medical Research Council Laboratories. Fajara, near Banjul, The Gambia Accepted for publication 29 January 1992 Summary Nine human monoclonal antibodies (MoAbs) recognizing 7 different antigenic structures of blood-stages of the human malarial parasite P.fakiparum (Pf) were produced by Epstein-Barr virus transformed B-cell lines (EBV-TCL) with or without fusion to the lymphoblastoid cell line KR4. The peripheral blood B-lymphocytes were obtained from 8 Gambian donors immune to Pf malaria. Two of the EBV-TCL could be expanded and maintained for more than 6months but neither one could be cloned. Six additional EBV-TCL were stabilized after fusion with the KR4 lymphoblastoid cell line. All resulting hybridomas permitted easy cloning. Some of the MoAbs produced distinct fluorescent staining patterns of asexual Pf blood-stage parasites when using high-resolution digitized videointensified fluorescence microscopy. Antigens on 195 kD and 155 kD proteins were. recognized by 3 and 1 MoAb, respectively, using Western blotting and immunoprecipitation techniques.
Keywords: Plasmodium falciparum, hybridoma; human monoclonal antibodies
The immune response to the human malarial parasite Plasmodium fakiparum (Pf) is mediated by both humoral and cellular reactions. Therefore, the identification and characterization of immunogenicPf blood-stage antigens (Ag) is one essential step before a protective immune response can be studied at the molecular level. Ideal reagents for the immunochemical characterization of Pf Ag would be human monoclonal antibody (MoAb) produced by B-lymphocytes from the immune human host. Several studies showed that MoAb to Pf antigens could be generated but that the yield of Ab producing Correspondence: Rupert Schmidt-Ullrich. $ Present address: Hoffmann-La Roche & Co., CH-4002 Basel, Switzerland. 8 Present address: National Institute for Medical Research, Division of Parasitology, The Ridgeway, Mill Hill, London NW7 IAA, UK.
45 1
452
M.Kamber et al.
cells was low and their stability is secreting Ab was limited (Fine et al. 1987, Lundgren et al. 1983, Schmidt-Ullrichet al. 1986, Udomsangpetch et al. 1986). The present study was undertaken in an attempt to define conditions for better sources of Pf-specific human MoAb. In comparingEpstein-Barr virus (EBV) transformed B-cells from donors immune to Pf and hybridomas derived from transformed B e l l s fused with the lymphoblastoid cell line KR4 (LCL; Kozbor et al. 1982) we examined their relative value as sources for high-yield and stable MoAb production. The human MoAbs produced were characterized in relation to Ag recognition by indirect immunofluorescence, immunoblotting, immunoprecipitation, and their activity in inhibiting Pf growth in vitro. Peripheral blood mononuclear cells (PBMC) from 8 Gambians donors, immune to Pf malaria with no recent episodes of acute malaria, were successfullytransformed with EBV (supernatant of mycoplasma-free 895-8 cells, 10 transforming units/cell; Miller & Lipman 1972) and subsequentlycultured according to a method described by Brown et al. 1986. These EBV-transformed cell lines (EBV-TCL) producing Ab against Pf antigens were subcultured by micromanipulation of individual cell clumps and finally expanded into T-25 tissue culture flasks. Mononuclear cells isolated from splenic tissue using FicollPaque gradients (Pharmacia LKB, Uppsala, Sweden), were obtained from an adult Gambian undergoing splenectomy and were transformed by EBV (SPL3B6). EBV-TCL from 7 donors were fused with the ouabain-resistant, HAT-sensitive LCL KR4 (Kozbor et al. 1982, obtained from Dr D. Kozbor) according to a previously reported protocol (Schmidt-Ullrich et al. 1986) with the addition of 10 p~ ouabain to the HAT selection medium. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (HyClone; Logan, UT), 4 mM glutamine, 1 mM Na-pyruvate, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.4 mM non-essential amino acids. Hybridomas were grown in the same medium supplemented with 0.0025 IU/ml bovine insulin and 2.5 p~ oxaloacetic acid. Cloning of EBV-TCL and hybridomas was performed by limited dilution at 0.3 cellslwell in flat-bottom microtitre plates with or without 2 x 105/mlhuman PBMC feeder cells irradiated to doses of 30 Gy. Cloning efficiency was defined as by Waldmann et al. (1987). Clonality of the cell lines was maintained through monthly recloning at the same stringency of 0.3 cellsfwell. The amount of secreted MoAb in culture supernatants was determined by ELISA using goat anti-human immunoglobulin (Ig) pre-coated on the bottom of 96-well plates and alkaline phosphatase-derivatized class- and IgG isotype-specific Ig (Shoenfeld et al. 1982).Of more than 50 EBV-TCLs produced from 8 donors (data not shown), only 8 cell lines from 7 donors were found to secrete Pf-specific Ab and could be expanded into T75 flasks to produce sufficient cell numbers for fusion with KR4 cells. With the exception of SS2F4, all EBV-TCLscould be stabilized after fusion with the KR4 cell line. Cloning was successful for all hybridomas but only for 4 of 8 EBV-TCL. However, clonability did not necessarily correlate with the stability of cell lines (Table I). With respect to MoAb production, the EBV-TCL invariably produced higher levels of Ab on the per cell basis than the corresponding hybridomas (Table 1). Considering the much improved growth properties of hybridomas the lower level of Ab production was easily compensated for by large scale cultures. Pf-specific Ab was characterized by indirect immune fluorescence using imaging technology (Mikkelsen et al. 1988), immunoblotting (Towbin et al. 1979), immunoprecipitation (Schmidt-Ullrichet al. 1986), or growth inhibition assay (Brown et al. 1986). All
Human MoAbs against P.falciparum
Table 1. Comparisons between EBV-transformed B-cell lines and their hybridomas on growth stability and antibody production
Cell line SS2F4 E*
Hi SS2D2 E H X-509 E H AJ-D2 ES H SPL3B6 E H HJ6D6 E H FS-G2 E H FS-E7 E
H
Stability (months)
Cloning efficiency (range %)
Ab production$ (pg/ml, range)
> 10 >8
n.c. 12-33
1 0-05-0.5
8
n.c. 2.2
1*3 0.15
8
47
6.3
4.8-33
0.5-1.9
>6
n.a. 13-20
n.a. n.d.
2.6 3-22
n.d. n.d.
>5
71 11-25
1.4 0.12
5 n.e.
2-1 n.a.
n.d. n.a.
79
n.e.
n.c. n.a.
n.d. n.a.
2 >6
n.c. 6-33
n.d. n.d.
4
