Screening of a Babesia bigemina cDNA Library with Monoclonal Antibodies Directed to Surface Antigens" J.
FIGUEROA,~G. M. BUENING,~v. MISHRA,~ AND T. F. McELWAIN bDepartment of Veterinary Microbiology ColIege of Veterinary Medicine University of Missouri-Columbia Columbia, Missouri 6S211 'Department of Infectious Diseases College of Veterinary Medicine University of Florida Gainesville, Florida 3261 1-0633
dl?epartment of Veterinary Microbiology and Pathology College of Veterinary Medicine Washington State University Pullman, Washington 99163
INTRODUCTION Bovine babesiosis, a disease of cattle caused by intraerythrocytic protozoan parasites of the genus Babesia, occurs in areas of the world in which the tick vector Boophilus sp. is continuously or sporadically present.' Of the species infecting cattle, Babesia bigemina has been considered less pathogenic than B. bovis; its prevalence in nature, however, is higher than that of B. bovis and disease outbreaks may result in important economic losses for the cattle industry.',2The clinical disease and pathology of babesiosis in cattle appear to be the direct result of the continuous asexual multiplication of the parasite within the host erythrocytes. Invasion and lysis of erythrocytes are manifested clinically as hemolytic anemia, hemoglobinuria, fever, hypoxia, and plasma protein abnormalities. The surface coat of the merozoite of Babesia has been demonstrated to elicit the production of antibodies that prevent erythrocyte invasion by the p a r a ~ i t e . ~ , ~ Previous studies also suggest that epitopes critical to the attachment of merozoites to bovine erythrocytes will be conserved within and among different parasite isolate^.^^^ Selection and characterization of candidate Babesia antigens for evaluation as immunogens have been hampered in the past because of the relatively low yields of parasite components obtained from in vitro or in vivo culture techniques. Large-scale production of Babesia antigenic components by modern molecular biology techniques through recombinant DNA would facilitate the selection, characterization, and definition of subunit parasite components with potential use as immunogens. This study describes 'a3
'Supported by USAID grant DAN-4178-A-00-7056-00. 122
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the preliminary characterization of recombinant clones expressing B. bigemina surfaceexposed epitopes.
MATERIALS AND METHODS Parasites An original Bubesiu bigeminu isolate from Mexico6 was established and grown continuously in vitro. JG-29,' a biologically cloned B. bigeminu line, was maintained in continuous culture under conditions slightly modified from those described prev i o u ~ l y Culture .~~ medium consisted of M-199 containing 40% (vol/vol) adult bovine serum, 20 mM TES, 100 units/mL of penicillin, 100 pg/mL streptomycin, and 0.25 pg/mL amphotericin B. Cultures were initiated in an incubator with gas concentration of 2% 0,, 5% CO,, and 93% N,, and maintained at 5% O,, 5 % CO,, and 90% N,.
Monoclonal Antibodies Hybridoma formation, selection, cloning, and characterization of monoclonal antibodies (mAb) against B. bigemina surface antigens have been previously reported.'.' Hybridomas secreting mAb 12C2 (IgG2a), mAb BlC9A2 (IgGl), mAb C2F3G3 (IgG2b), mAb BlB3C4 (IgG2a), mAb BlE2E8 (IgM), and mAb FoxG5 (IgG2a) were grown to confluence (4-5 days) in RPMI 1640 medium containing 10% fetal calf serum in a 5% 0,, 5% CO,, and 90% N, atmosphere at 37 "C. Culture supernatants from several 75 cm2 culture flasks of each hybridoma were collected, centrifuged at 3,953 g for 10 min, and filter-sterilized through a 0.2-pm pore diameter membrane.
Parasite Antigen Solubilization Bubesiu bigeminu infected erythrocytes from in vitro cultures were concentrated to 90-95% parasitemia as described previously.12 After glycerol-enhanced osmotic shock,I3the B. bigemina-infected erythrocyte ghosts were extracted for 1 h on ice with five volumes of extraction buffer7,I4(PBS, pH 8.0; 5 mM iodoacetamide; 2mM PMSF; 5 mM EDTA; 2 mM sodium metabisulfite; 17 mU aprotinin; 1% NP-40; and 0.1% sodium deoxycholate). After centrifugation at 13,600 g for 15 min at 4 "C the extract supernatant was recovered and frozen at -70 "C. Normal bovine erythrocytes were also processed as above and the protein extract was included as a control for the immunoassays. Protein content was determined as previously described. I s
Construction of AZAP Expression Library, Immunoscreening, and Phagemid Excision The procedures utilized in the construction of a B. bigemina cDNA library have been described in detail elsewhere.' Briefly, poly A+ mRNA purified from the B. bigemina JG-29 clone was used to synthesize cDNA according to published proceduresL6with minor modifications. Double-stranded cDNA with EcoRI adaptors was ligated to AZAPII EcoRI-digested vector arms and packaged into Aphage heads using Gigapack Gold extracts as recommended by the supplier (Stratagene Cloning Systems,
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La Jolla, CA). The resulting library was plated on the E. coli cell line PLK-F, and a 2 X lo6 plaque-forming portion of the amplified library (phage lysate containing units) was prepared for this study. The cDNA library was screened with pooled mAb-containing culture supernatant at a 1:6 final dilution. The plaque immunoassay procedure was performed using a picoBlueTMimmunoscreening kit and E. coli XL 1-Blue host cells (Stratagene). Positive plaques were eluted in 1 mL of SM buffer (100 mM NaCI; 50 mM Tris, pH 7.5; 10 mM MgSO,; 0.01% [wt/vol] gelatin),” and the recombinant phages were subjected to a second and third round of plaque immunoscreening to insure purity of the recombinant. In addition, the recombinants were plaque-immunoscreened individually with each mAb. The Bluescript SK (-) (Stratagene) phagemid sequences positioned in hZAP recombinant phageI8were excised in vivo essentially as describedI8in the supplier’s instruction manual (Stratagene).
Recombinant pBluescript Purification and Restriction Enzyme Digestion
E. coli XL1-Blue cells carrying the phagemid of interest were subjected to the alkaline lysis method and the phagemids were purified as described before.” Purified plasmid DNA (1 pg) from the recombinant phagemid or pBluescript vector alone were single-digested with EcoRI (10 units) or double-digested with Sac1 and XhoI (10 units each) restriction enzymes (Promega Corporation, Madison, WI) with an overnight incubation at 37°C in the appropriate buffer. The digestion products obtained were analyzed by horizontal gel electrophoresis in a 0.8% (wt/vol) agarose gel in 0.045 M Tris-borate, 0.001 M EDTA buffer containing 0.1 pg/mL of ethidium br0mide.I’ Immunoblotting of B. bigemina Proteins Expressed in Recombinant Phagemids XL1-Blue cells containing the recombinant pBluescript were grown as suggested by the supplier (Stratagene) with or without the addition of isopropyl-B-thiogalactopyranoside (IPTG) to the cells (10 mM final concentration). Cultures were grown until just reaching stationary phase (- O.D., = 1.0). The cells were then centrifuged at 1,600 g for 15 min and the pellets resuspended in lysis buffer (50 mM Tris-HC1, pH 8.0; 1 mM EDTA; 1 FM PMSF; 10% [wt/vol] sucrose) to which lysozyme was added to 1 mg/mL final concentration. After incubation for 10 min on ice, Nonidet P-40 was added to 0.1 % final concentration and further incubated for 10 min on ice. The lysates were centrifuged at 12,000g for 1 h and the supernatants were recovered and stored at - 70 “C until immunoblotting analysis was performed. Protein determination was done as previously reported. Is Lysate samples of recombinant phagemid (25 pg) were added to an equal volume of Laemmli sample buffer (0.125 M Tris-HC1, pH 6.8; 6% SDS; 20% glycerol; 10% P-mercaptoethanol; 0.1%bromophenol blue) and boiled for 5 min before loading the sample into a discontinuous polyacrylamide gel (4% stacking gel, 10% separating gel). Gel electrophoresis was carried out at 60 mAMP using the buffers of Laemmli.‘’ The electrophoretic transfer of polypeptide bands to nitrocellulose was performed as described previously.20Detection of B. bigemina epitopes expressed as fused polypeptides with P-galactosidase by the recombinant pBluescript phagemids was carried out using mAb B 1B3C4 and mAb C2F3G3 (immunoglobulin concentration, 5 pg/mL). In addition, an mAb to B. bigemina (FoxG5, IgG2a) that recognizes different polypeptides was included in the immunoassay as a control antibody.
FIGUEROA et 01.: SCREENING OF B. BICEMIA'A cDNA LIBRARY
FIGURE 1. (A) Phagemids rescued from AZAP bacteriophage recombinant clones by the in vivo excision procedure using helper phage R408.(B) Restriction enzyme digests of rescued phagemids of recombinant AZAP Bbi clones. Lane 1, Sk(-), phagemid vector rescued from nonrecombinant AZAP. Lanes 2 to 6, AZAP Bbi recombinant phagemids rescued from recombinant AZAP Bbi 1-5 clones. Lane M, Hind11 digested ADNA markers: 23,130; 9,416; 6,557; 4,361; 2,322; 2,027; 564 Bp.
RESULTS The six mAb utilized in this study bind to surface-exposed epitopes on B. bigemina parasites as evidenced by an indirect fluorescent antibody test with live parasites or by immunoelectron microscopy.' The mAb pool did not cross-react with E. coli proteins nor did they bind to normal erythrocyte components at a 1:6 dilution. Initially, 3 x lo5PFU from the amplified library were immunoscreened with the pooled mAb. Five plaque-lift positive clones were identified (AZAP Bbil, 2, 3, 4, and 5) and phagemids rescued by in vivo excision as described. When tested individually with each of the mAb, all AZAP Bbi clones were reactive with mAb C2F3G3 and mAb BlB3C4. Purification of recombinant phagemid and analysis of undigested DNA by agarose gel electrophoresis indicated the presence of cDNA inserts with different sizes (FIG. 1A). To determine the size of the insert in the recombinant phagemid, DNA was digested with EcoRI and the fragments obtained analyzed by agarose gel electrophoresis (FIG. 1B). The results indicated that AZAPBbi1 carried a cDNA insert of approximately 600 base pairs (Bp). AZAPBbi2, AZAPBbi3, and AZAPBbi5 carried an insert that contained an internal EcoRI site, producing two fragments of approximately 1.1 kBp and 0.6 kBp (AZAPBbi2 and AZAPBbiS) or 1.05 kBp and 0.65 kBp (AZAPBbi3). Restriction enzyme digests of recombinant phagemids with Sac1 and XhoI (enzymes with recognition sites flanking the EcoRI site where the cDNA was inserted) demonstrated a cDNA insert of 1.7 kBp present in AZAPBbi2, AZAPBbi3, and AZAPBbi5.
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AZAPBbi1 contained either a Sac1 or a XhoI site in the insert as evidenced by the presence of the two smaller fragments (-300 Bp and 200 Bp) (FIG. 1B). Immunoblotting analysis of protein extracts from XL1-Blue cells containing the recombinant phagemids showed that both mAb C2F3G3 and mAb BlB3C4 bound primarily to a polypeptide of 55 kDa, being expressed by clones AZAPBbi2, AZAPBbi3, and AZAPBbi5 (FIG.2). The 55 kDA recombinant polypeptide was expressed in clones hZAPBbi3 and AZAPBbi5 whether or not the cells were induced with IPTG, whereas AZAPBbi2 expressed the 55 kDa polypeptide only after IPTG induction (FIG. 2A and B). The recombinant polypeptide migrated slightly faster than the native B. bigemina antigens recognized by mAb C2F3G3 and mAb BlB3C4 (60 and 58 kDa). mAb C2F3G3 and mAb BlB3C4 also bound to smaller polypeptides of 36.5 kDa, 32 kDa, and 30 kDa apparent size. All recombinant polypeptides were specificallydetected by mAbs C2F3G3 and BlB3C4 as mAb FoxG5, the immunoglobulin isotype control; at the same concentration it did not bind to any of the polypeptides in the E. coli cell protein lysate but bound to native B. bigemina antigens of -43 kDa and 36 kDa (FIG. 2C).
DISCUSSION Because of the renewed interest in developing an immunogen capable of preventing the clinical manifestations of the disease caused by B. bigemina in cattle, efforts are being directed toward the identification, characterization, and immunogenic (protective) analysis of parasite surface antigens with potential use as immuno25 By the utilization of modern molecular biology techniques and procegen~."',~,'','~.~'dures through the manipulation of recombinant DNA, important parasite components can now be produced in large amounts to be tested and utilized as protective immunogens or immunodiagnostic reagent^.*^-^^ In this study we tested six mAb to B. bigemina that recognize surface-exposed epitopes in the erythrocytic stages of the para~ite.~ Immunoscreening of a cDNA library constructed in the vector AZAP'O with the mAb to B. bigemina has resulted in the isolation to five recombinant clones. All five clones reacted with mAb BlB3C4 and mAb C2F3G3. The inserts present in AZAPBbi2, AZAPBbi3, and AZAPBbi4 appeared to have a similar size and the presence of similar restriction enzyme recognition sites. Whether these cDNA inserts belong to the same gene or share sequence homology will not be known until cross-hybridization studies are carried out both between each other and between the cDNA insert and genomic B. bigemina DNA. However, that AZAPBbi2, AZAPBbi3, and AZAPBbi5 clones actually carry a very similar cDNA insert was demonstrated by the common recognition with mAb BlB3C4 and mAb C2E3G3 of a 55 kDa polypeptide that had the same apparent mobility in all three recombinant E. coli lysates. It was previously reported that mAb C2F3G3 bound strongly to B. bigemina components of 62 kDa, 58 kDa, and weakly to 49 kDa.6 The 9
FIGURE 2. Immunoblot analysis of babesia bigemina surface protein expression from pBluescript phagemid. (A) Reaction with mAb JC2F3G3. (B) Reaction with mAb BlB3C4. (C) Reaction with mAb FoxG5. Lane 1, protein lysate from XL1-blue E. coli cells. Lane 3, XLI-Blue with pBluescript. Lanes 5, 7, 9, 11, and 13, JXLI-blue with recombinant pBluescript from hZAP Bbi 1-5 clones, respectively. Lanes 2, 4, 6, 7, 10, 12, and 14, protein lysates of I, 3, 5, 7, 9, 11, and 13, respectively, after IPTG induction. Lanes 15 and 16, B. bigemina-infected erythrocyte protein lysate at 50 pg and 25 pg, respectively. Lanes 17 and 18, bovine normal erythrocyte protein lysate at 50 pg and 25 pg, respectively.
FIGUEROA et aL: SCREENING OF B. BIGEMINA cDNA LIBRARY
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same components were recognized by mAb BlB3C4 in addition to antigens with molecular sizes of 47-5 1 kDa. Although recognizing B. bigemina components of similar size, however, mAb C2F3G3 and mAb BlB3C4 apparently detect different epitopes on these components as evidenced by the distinct staining patterns using the indirect fluorescent antibody test on acetone-fixed parasites. I" Experimentally infected cattle produced relatively high antibody levels to these parasite antigen^.'^ The antibody was still detectable 8 months postinoculation. Additionally, parasite components detected by mAb C2F3G3 and mAb BlB3C4 are commonly present in B. bigemina isolates from North and Central America and the Caribbean.'.I4 The antigens also appear to have neutralization-sensitive epitopes as evidenced by the decreased in vitro multiplication of B. bigemina exposed to mAb C2F3G3 and BIB3C4.l' B. bigemina polypeptides with a similar molecular weight to those recognized by mAb C2F3G3 and BlB3C4 have been previously identified with another set of mAb to B. bigemina. 6,9 The parasite polypeptide immunoprecipitated by mAb C2F3G3 (57 kDa), however, was shown to be smaller in size to that immunoprecipitated by mAb 14/16.1.7 (58 kDa) when the two mAb-immunoprecipitated 35S-labeledantigens were analyzed side by side in SDS-PAGE (T. F. McElwain, unpublished observation). Polypeptide p58 also contains a neutralization-sensitive epitope, is a surface protein, and is antigenically conserved among B. bigemina isolate^.^ A recombinant clone encoding p58 has been sequenced and recombinant p58 protein has been expressed.' Further comparison of the genes encoding both p58 and the clones identified in this study will clarify whether these antigens are related gene products. The difference in size of the largest B. bigemina polypeptide expressed in E. coli and recognized by the mAb C2F3G3 and BlB3C4 with those expressed naturally by the parasite could be an indication that the B. bigemina cDNA insert contained in the recombinant phagemids is not a full-length copy of the parasite gene encoding the surface antigens. Alternatively, the recombinant polypeptide described in this study could be encoded by one of a family of B. bigemina genes encoding polypeptides with surface-exposed epitopes recognized by parasite-specific mAb.' The smaller polypeptides detected by mAb C2F3G3 and BlB3C4 in the protein lysate of the recombinants are, perhaps, degradation products of polypeptide 5 5 kDa. But it is also possible that the various polypeptides bound by mAb BlB3C4 and mAb C2F3G3 are part of a large protein complex. We are in the process of defining the relationship of these polypeptides. Sequencing and analysis of the cDNA inserts present in the recombinant clones will clarify some of the issues decribed above.
SUMMARY A Babesia bigemina cDNA library prepared in hZAP bacteriophage vector was immunoscreened to detect clones expressing surface-exposed epitopes of B. bigemina. A nonradioactive indirect plaque-lift immunoassay was used to detect the positive clones. The primary antibody consisted of a pooled sample of six monoclonal antibodies (mAb) specific for B. bigemina that recognizes various parasite surface antigens of different molecular mass. Screening of approximately 300,000 plaque-forming units from the hZAP cDNA expression library resulted in the identification of five positive clones. The five recombinant clones were immunoscreened individually with each of the six mAb. All five independently obtained clones consisted of hZAP recombinants expressing B. bigemina components recognized by mAb C2F3G3 and B 1B3C4. Restriction enzyme digests of rescued recombinant phagemids showed that only four clones contained B. bigemina cDNA. One clone @ZAP Bbil) contained an insert of approxi-
FIGUEROA el ul.: SCREENING OF B. BIGEMINA cDNA LIBRARY
mately 0.6 kBp whereas the other three clones (AZAP Bbi2, AZAP Bbi3, and AZAP Bbi5) carried a cDNA insert of approximately 1.7 kBp. Immunoblotting of protein extracts from recombinants AZAP Bbi2, AZAP Bbi3, and AZAP Bbi5 with mAb C2F3G3 and B1B3C4 demonstrated the expression of a recombinant B. bigemina polypeptide of 55 kDa in E. coli. ~ O T E DNA : sequencing of clone AZAP Bbi-3 revealed an open reading frame 1,440 residues long, whose nucleotide sequence was homologous to a previously published Babesia bigemina DNA sequence that codes for p58 surface protein.']
ACKNOWLEDGMENTS We thank Karen McLaughlin for technical assistance and Ellen Swanson for typing the manuscript. REFERENCES 1. CALLOW,L. L. 1984. Piroplasms. In Animal Health in Australia: Protozoal and Rickettsia1 Diseases. Vol. 5 121- 167. Bureau of Animal Health, Australian Government Publishing Service. Canberra. 2. MCCOSKER,P. J. 1981. The global importance of babesiosis. In Babesiosis. M. Ristic & J. P. Kreier, Eds.: 1-24. Academic Press. New York, NY. D. F. 1979. Babesiosis. In Proceedings No. 42 of the J. D. Stewart Memorial 3. MAHONEY, Refresher Course in Cattle Diseases. University of Sydney. Sydney, Australia. 4. SMITH,R. D. & M. RISTIC. 1981. Immunization against bovine babesiosis with culturederived antigens. In Babesiosis. M. Ristic & J. P. Kreier, Eds.: 485-507. Academic Press. New York, NY. 5 . MCELWAIN,T. F., L. E. PERRYMAN, A. J. MUSOKE& T. C. MCGUIRE.1991. Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins. Mol. Biochem. Parasitol. 47: 213-222. 6. MCELWAIN,T. F., L. E. PERRYMAN, W. C. DAVIS& T. C. MCGUIRE.1987. Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites. J. Immunol. 138: 2298-2304. 7. FIGUEROA, J. V., G. M. BUENING,D. A. KINDEN& T. J. GREEN.1990. Identification of common surface antigens among Babesia bigemina isolates by using monoclonal antibodies. Parasitology 100 161-175. 8. VEGA, C. A., G. M. BUENING,T. J. GREEN& C. A. CARSON.1985. In vifro cultivation of Babesia bigemina. Am. J. Vet. Res. 46 416-420. 9. MISHRA,V. F., E. B. STEPHENS,J. B. DAME,L. E. PERRYMAN, T. C. MCGUIRE& T. F. MCELWAIN.1991. Immunogenicity and sequence analysis of recombinant p58-A neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface protein. Mol. Biochem. Parasitol. 47: 207-212. 10. VEGA,C. A., G. M. BUENING,S. D. RODRIGUEZ & C. A. CARSON.1986. Cloning of in vifro propagated Babesia bigemina. Vet. Parasitol. 2 2 223-233. 11. FIGUEROA, J. V. & G. M. BUENING.1991. In vitro inhibition of multiplication of Babesia bigeminu by using monoclonal antibodies. J. Clin. Microbiol. 29(5): 997- 1003. 12. VEGA,C. A., G. M. BUENING,S. D. RODRIGUEZ & C. A. CARSON.1986. Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes. J. Protozool. 33: 514-518. 13. FIGUEROA, J. V., G. M. BUENING& D. A. KINDEN.1990. Purification of the erythrocytic stages of Bubesiu bigemina from cultures. Parasitol. Res. 7 6 675-680. 14. FIGUEROA, J. V., G. M. BUENING, R. HERNANDEZ, M.A. MONROY,J. A. RAMOS,G. J. CANTO,J. A. ALVAREZ& C. A. VEGA. 1989. Evaluation of the serologic response of calves experimentally infected with Babesia bigeminu by ELISA and immunoblotting
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