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Expression, purification and characterization of a functional region of the Plasmodium vivax Duffy binding protein
Molecular and Biochemical Parasitology 109 (2000) 179 – 184 www.elsevier.com/locate/parasitology
Short communication
Expression, purification and characterization of a functional region of the Plasmodium 6i6ax Duffy binding protein Sheetij Dutta, Jon R. Daugherty 1, Lisa A. Ware, David E. Lanar, Christian F. Ockenhouse * Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant A6enue, Sil6er Spring, MD 20910, USA Received 22 December 1999; received in revised form 30 March 2000; accepted 6 April 2000
Keywords: Malaria; Duffy binding protein; Baculovirus expression; Plasmodium 6i6ax
Erythrocytes of Duffy-negative individuals are resistant to invasion by the parasite Plasmodium 6i6ax [1]. A 140-kDa protein, the Duffy binding protein (PvDBP), has been identified as the parasite ligand for Duffy-glycoprotein receptor on human erythrocytes [2,3]. PvDBP gene sequence analysis [4] reveals that it belongs to a family of Plasmodium adhesion proteins found in several Plasmodia including the P. falciparum erythroAbbre6iations: Bv-PvDBPrII, baculovirus recombinant PvDBPrII protein; IL-8, interleukin-8; PvDBPrII, region II of P. 6i6ax Duffy binding protein. Disclaimer: The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. * Corresponding author. Tel.: + 1-301-3199473; fax: + 1301-3199012. E-mail address: [email protected] (C.F. Ockenhouse). 1 Present address: Division of Vaccines and Related Products Applications, OVRR¯CBER¯FDA, Rockville MD, USA.
cyte-binding antigen (EBA-175) and the variant surface antigen PfEMP-1 encoded by 6ar genes [5–7]. In the N-terminal region of PvDBP, a 324 amino acid cysteine-rich stretch or ‘region II’ (PvDBPrII), has been identified as the principal erythrocyte binding domain using transfected COS cells [8,9], making PvDBPrII an attractive vaccine candidate. Substantial quantities of correctly folded PvDBPrII protein are needed to initiate structural and immunological studies. We have expressed PvDBPrII gene in Escherichia coli system; however, the protein is contained predominantly within the insoluble fraction (unpublished observation). The E. coli product did not show appreciable erythrocyte binding activity, similar to a previous report with E. coli-expressed region II-containing construct [10]. In addition, there are no conformation-specific monoclonal antibodies to analyze the structure of refolded PvDBPrII protein from E. coli. Hence, we have focused on the eukaryotic baculovirus system for production
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of PvDBPrII. Here we present the expression, purification and characterization of recombinant PvDBPrII protein produced in the insect cell system. PCR primers, forward: 5%-atgcgcggccgctACGATCTCTAGTGCTATTATA-3% and reverse: 5%atatgaattcTGTCACAACTTCCTGAGTATT-3% (restriction sites shown in lower case), were used to amplify PvDBPrII encoding gene (amino acid residues 198–521; TISSAII…to…NTQEVVT) from genomic DNA of Salvador I strain of P. 6i6ax. PCR product was cloned into the Not I –EcoR I sites of baculovirus transfer plasmid pBSV-8His [11] in-frame with a vector encoded
human complement regulatory factor H-like 1 plasma protein (FHL-1) signal peptide on the amino-terminus (Fig. 1A). The construct also encodes an enterokinase cleavage site followed by a 8× histidine tag at the carboxy-terminus (Fig. 1A). The vector pBSV-8His is designed to direct expressed proteins to a secretory pathway and allow easy purification of the product from culture supernatant of insect cells [12]. The cloned insert was evaluated by DNA sequencing, and recombinant PvDBPrII baculovirus clones were generated essentially as described in the Clontech Laboratories BacPAK6 manual (Palo Alto, CA). Cloned virus stock was amplified from a single plaque.
Fig. 1. Expression and purification of the recombinant Bv-PvDBPrII construct. (A) Schematic view of the PvDBP gene (top) showing the conserved amino acid blocks with other EBPs; shaded areas are N- and C-terminal cysteine-rich regions (N-, C-cys); transmembrane (TM). Expressed Bv-PvDBPrII construct (bottom) shows the extra residues encoded by vector pBSV-8His; (*) indicates translation stop. (B) Coomassie-stained SDS-PAGE analysis of Bv-PvDBPrII during various stages of purification. Lane 1, 6.5× concentrated baculovirus culture supernatant; 2, flow-through of Ni2 + column; 3, pooled fractions containing BvPvDBPrII eluted off Ni2 + column and loaded on SP Sepharose; 4, flow-through of the SP Sepharose column; 5, 1 M NaCl eluate of SP Sepharose containing pure Bv-PvDBPrII protein indicated by the arrow. Molecular weight in kDa indicated on left.
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The 367 amino acid long Bv-PvDBPrII polypeptide has a predicted molecular weight of 41 kDa after FHL-1 signal peptide cleavage. Expressed Bv-PvDBPrII secreted into the culture supernatant migrated close to 40 kDa on SDSPAGE as detected by western blot with anti-6× histidine monoclonal antibody (data not shown). The Bv-PvDBPrII appeared as a compact band on non-reducing gels confirming that the majority is present as a single conformer. Its mobility on SDS-PAGE was decreased upon reduction with DTT confirming its disulphide bonded nature, and the protein did not seem to undergo extensive glycosylation in the baculovirus system as assessed by staining the SDS-PAGE with the GelCode® Glycoprotein Staining Kit (Pierce, Rockford, IL) (data not shown). N-terminus amino acid sequencing of purified Bv-PvDBPrII protein revealed the first six amino acids as Thr – Ile – Ser–Ser–Ala–Ile which corresponds to the first six residues of native PvDBPrII, confirming that the signal peptide was efficiently cleaved during secretion. We found it unnecessary to cleave the 8-histidine tag at the C-terminus, as it had no effect on erythrocyte binding ability of BvPvDBPrII. Recombinant Bv-PvDBPrII was produced in large scale using a Bellco Bioreactor with a 10-l working volume. Sf21 insect cells were grown, in serum-free medium (Cyto-SF9, Kemp Biotechnologies, Frederick, MD), culture was maintained at 27°C and the dissolved oxygen level was at 60% of air saturation. At a density of 1.5× 106 cells ml − 1 the culture was infected with recombinant PvDBPrII baculovirus. Seventy-two hours after infection, supernatant was harvested and concentrated at 4°C using a 10 000 MWCO hollow-fiber ultrafilter (A/G Technology, Woburn, MA) to 1.5 l (a 6.5-fold reduction in volume). The concentrated supernatant was aliquoted and stored at −80°C. Bv-PvDBPrII constituted about 8% of total protein in the 6.5-fold concentrated baculoviral culture supernatant (Fig. 1B, lane 1) as estimated by densitometric analysis of Coomassie stained SDS-PAGE gels. Highly purified fractions of the PvDBPrII were obtained using a two-step purification protocol which was carried out at room temperature. A
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typical purification run was started with 50 ml of concentrated supernatant, it was cleared by centrifugation at 10 000× g for 15 min and 5 ml of 10× PBS (1.5 M NaCl, 17 mM KH2PO4, 50 mM Na2HPO4, pH 7.4) was added to it. The supernatant was passed over a 5-ml Ni-NTA SUPERFLOW column (Qiagen; Valencia, CA), on a FPLC system (Waters, MILLIPORE; Milford, MA). The column was washed extensively with 1× PBS followed by PBS containing 500 mM NaCl and 40 mM imidazole. Bv-PvDBPrII was eluted with a 40 to 200 mM imidazole linear gradient in 50 mM Tris, 0.05% Triton X-100 (pH 7.0). Triton X-100 enhanced the recovery and purity during the second step of purification. The first step of purification on Ni2 + column resulted in up to 63–73% pure protein fractions (Fig. 1B, Lane 3). Elution fractions containing BvPvDBPrII were pooled, diluted fourfold in the same buffer without imidazole, and loaded on a 1-ml SP Sepharose Fast Flow cation exchange column (Amersham–Pharmacia Biotech; Piscataway, NJ). The column was washed with 50 mM Tris buffer containing 100 mM NaCl (pH 7.0) and purified Bv-PvDBPrII was eluted in 50 mM Tris (pH 7.0) containing 1 M NaCl. Eluted protein was dialyzed against 1× PBS overnight and stored at −20°C. Estimated purity of the final product was 92–96% (Fig. 1B, Lane 5). An average of 2 mg Bv-PvDBPrII protein was purified per liter of the baculovirus supernatant. Purified Bv-PvDBPrII protein was analyzed further for its biological activity using an erythrocyte binding assay (see Fig. 2A legend). BvPvDBPrII protein bound Duffy-positive human erythrocytes (Fig. 2A) while no binding was observed with Duffy-negative erythrocytes. Positive binding was also shown with Aotus monkey erythrocytes, but not with Rhesus monkey erythrocytes. This data corroborates the observed binding properties for native PvDBP from P. 6i6ax culture supernatant [2]. In order to evaluate the specificity of this interaction we pretreated human Duffy-positive erythrocytes with either trypsin or chymotrypsin, and assayed these treated erythrocytes for Bv-PvDBPrII-binding activity. Chymotrypsin which is known to remove the Duffy antigen from erythrocytes abolished the
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Fig. 2. Biological and immunological characterization of BvPvDBPrII protein. (A) Erythrocyte binding assays were performed using Aotus, Rhesus, and human erythrocytes (Duffy-positive (pos), Duffy-negative (neg)). Erythrocytes (12 ml of 50% hematocrit) in PBS were added to 500 ml PBS containing 0.1% BSA along with 1 mg Bv-PvDBPrII protein and incubated at 37°C for 30 min. Erythrocytes were washed twice with 0.5 ml PBS and bound protein eluted in 30 ml 1 M NaCl containing 1 mM PMSF. Eluted protein was detected on a western blot immunostained with affinity purified polyclonal anti-Bv-PvDBPrII as the primary antibody. The blot was developed with SuperSignal Chemi-luminescent substrate reagent (Pierce) and exposed on Kodak X-Omat Blue XB-1 film (Eastman Kodak, Rochester, NY). (B) Effect of trypsin or chymotrypsin enzyme treatment on Bv-PvDBPrII binding to human Duffy-positive erythrocytes. (C) Competitive inhibition of Bv-PvDBPrII binding to human Duffy-positive erythrocytes with 0, 0.25 and 2.5 mM IL-8. (D) Recognition of Bv-PvDBPrII by human immune sera in immunoblot (serum dilution 1:1000; top panel). Inhibitory activity of Bv-PvDBPrII binding to Duffy-positive red cells using the same set of sera (bottom panel). (E) Indirect immunofluorescence assay, on methanol fixed Aotus erythrocytes infected with P. 6i6ax Sal I strain, with affinity purified 20 mg ml − 1 rabbit anti-Bv-PvDBPrII antibodies. Fluorescence image of a mature schizont (arrow) containing individual merozoites appearing as dots (magnification × 1000).
binding of Bv-PvDBPrII to Duffy-positive erythrocytes (Fig. 2B). Trypsin treatment which has no effect on the Duffy antigen did not cause any reduction in Bv-PvDBPrII binding to erythrocytes. Recombinant IL-8 has been shown to compete with PvDBPrII for binding to the Duffy antigen on erythrocytes [8,13]. Inhibition of Bv-PvDBPrII binding to human Duffy-positive erythrocytes was done by pre-incubating erythrocytes with two concentrations of recombinant human IL-8 (R&D systems; Minneapolis, MN) for 30 min at 37°C followed by erythrocyte binding assay. IL-8 at 0.25 mM concentration inhibited approximately 75% Bv-PvDBPrII binding (Fig. 2C) and close to 90% inhibition was observed at 2.5 mM estimated by densitometer. In order to assess whether the protein was antigenic, human immune sera collected from adults attending a malaria clinic on the Thai– Burmese border as part of a larger study examining immune responses to P. falciparum and P. 6i6ax antigens (data to be presented elsewhere) were tested by immunoblot for reactivity to recombinant Bv-PvDBPrII. A preliminary examination indicated that PvDBPrII was indeed antigenic, in that the protein reacted to antibodies present in some serum samples (Fig. 2D, lanes 1,2,4,6 top panel). Furthermore, these same serum samples were also analyzed for their ability to block Bv-PvDBPrII binding to human Duffy-positive erythrocytes. Binding inhibition was done by incubating 1 mg Bv-PvDBPrII with 10 ml of human serum (final dilution 1:5) for 1 h at room temperature prior to its addition to the erythrocyte suspension (Fig. 2D, bottom panel). Although some of the sera showed the presence of erythrocyte binding-inhibitory antibodies (lanes 2 and 6, bottom panel), other samples (lanes 1 and 4, bottom panel) had no detectable anti-PvDBP blocking activity despite being highly reactive to PvDBPrII on immunoblot. Further immunologic characterization indicated that Bv-PvDBPrII is immunogenic in addition to being antigenic. Anti-PvDBPrII antibodies affinity-purified from rabbit serum immunized with Bv-PvDBPrII (in Freund’s adjuvant), reacted with merozoites within late stage schizonts on
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indirect immunofluorescence assay (Fig. 2E), similar to the pattern observed previously in P. knowlesi schizonts [14], further establishing the near native structure of the Bv-PvDBPrII. Plasmodium antigens, such as the PvDBPrII, containing a large number of cysteine and aromatic amino acids pose a problem of insolubility during expression in the E. coli system. Although the total yield of PvDBPrII produced in E. coli is considerably larger than baculovirus, the fraction of correctly folded and functional molecules is low as indicated by poor erythrocyte binding characteristics (Dutta et al., unpublished data). The baculovirus system offers several advantages over prokaryotic systems including secretion of the expressed protein into the culture supernatant, correct folding, signal peptide cleavage and posttranslational modifications. Bv-PvDBPrII expressed in baculovirus system showed biological characteristics similar to those reported for native P. 6i6ax PvDBP protein from culture supernatant or COS cell expressed region II. Immunological characteristics of Bv-PvDBPrII confirms the near native structure of recombinant protein. Human immune sera from individuals residing in an endemic area not only recognized Bv-PvDBPrII on western blot and ELISA (data not shown), but in some cases were able to block Bv-PvDBPrII binding to erythrocytes. Interestingly, the presence of high titer antibodies to recombinant PvDBPrII did not necessarily correlate with its ability to block erythrocyte binding in a preliminary analysis of persons exposed to and infected with P. 6i6ax. Further investigation is required to examine the nature of epitopes which elicit inhibitory antibodies and strategies are needed to optimize the production of inhibitory antibodies. Although it is possible that the activity observed in these in vitro erythrocyte binding assays may not reflect all of the possible anti-parasite effects present in vivo, and because of the limitations of in vitro culture of P. 6i6ax, the significance of these results await confirmation in the in vivo primate models of P. 6i6ax. Nevertheless, the baculovirus-expressed Duffy binding protein is an attractive vaccine candidate and plans are underway to immunize
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non-human primates with Bv-PvDBPrII and challenge with P. 6i6ax in the near future.
Acknowledgements We thank the National Research Council (Washington, DC) for funding S. Dutta; Peter F. Zipfel at Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany for the plasmid; Gregory E. Garcia and Deborah R. Moorad at WRAIR for N-terminal sequencing; Connie L. Howard at the Walter Reed blood bank; Christopher W. Kemp at Kemp Biotechnologies for fermentation; Patrick E. Duffy and V. Ann Stewart at WRAIR for P. 6i6ax parasites; David C. Miles at WRAIR for photography.
References [1] Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance factor to Plasmodium 6i6ax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med 1976;295:302 – 4. [2] Wertheimer SP, Barnwell JW. Plasmodium 6i6ax interaction with the human Duffy blood group glycoprotein: identification of a parasite receptor-like protein. Exp Parasitol 1989;69:340 – 50. [3] Barnwell JW, Nichols ME, Rubinstein PJ. In vitro evaluation of the role of the Duffy blood group in erythrocyte invasion by Plasmodium 6i6ax. J Exp Med 1989;169:1795– 802. [4] Fang XD, Kaslow DC, Adams JH, Miller LH. Cloning of the Plasmodium 6i6ax Duffy receptor. Mol Biochem Parasitol 1991;44:125 – 32. [5] Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH. A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci USA 1992;89:7085 – 9. [6] Okenu DM, Malhotra P, Lalitha PV, Chitnis CE, Chauhan VS. Cloning and sequence analysis of a gene encoding an erythrocyte binding protein from Plasmodium cynomolgi. Mol Biochem Parasitol 1997;89:301 – 6. [7] Peterson DS, Miller LH, Wellems TE. Isolation of multiple sequences from Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding protein. Proc Natl Acad Sci USA 1995;92:7100 – 4. [8] Chitnis CE, Miller LH. Identification of the erythrocyte binding domains of Plasmodium 6i6ax and Plasmodium knowlesi proteins involved in erythrocyte invasion. J Exp Med 1994;180:497 – 506.
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[9] Ranjan A, Chitnis CE. Mapping regions containing binding residues within functional domains of Plasmodium 6i6ax and Plasmodium knowlesi erythrocyte-binding proteins. Proc Natl Acad Sci USA 1999;96:14067–72. [10] Fraser T, Michon P, Barnwell JW, Noe AR, Al-Yaman F, Kaslow DC, Adams JH. Expression and serologic activity of a soluble recombinant Plasmodium 6i6ax Duffy binding protein. Infect Immun 1997;65:2772–7. [11] Kuhn S, Zipfel PF. The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins. Gene 1995;162:225 –9. [12] Prodinger WM, Schoch J, Schwendinger MG, Hellwage J,
Parson W, Zipfel PF, Dierich MP. Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector. Immunopharmacology 1997;38:141 – 8. [13] Horuk R, Chitnis CE, Darbonne WC, Colby TJ, Rybicki A, Hadley TJ, Miller LH. A receptor for the malarial parasite Plasmodium 6i6ax: the erythrocyte chemokine receptor. Science 1993;261:1182– 4. [14] Adams JH, Hudson DE, Torii M, Ward GE, Wellems TE, Aikawa M, Miller LH. The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 1990;63:141 – 53.
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Short communication
Expression, purification and characterization of a functional region of the Plasmodium 6i6ax Duffy binding protein Sheetij Dutta, Jon R. Daugherty 1, Lisa A. Ware, David E. Lanar, Christian F. Ockenhouse * Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant A6enue, Sil6er Spring, MD 20910, USA Received 22 December 1999; received in revised form 30 March 2000; accepted 6 April 2000
Keywords: Malaria; Duffy binding protein; Baculovirus expression; Plasmodium 6i6ax
Erythrocytes of Duffy-negative individuals are resistant to invasion by the parasite Plasmodium 6i6ax [1]. A 140-kDa protein, the Duffy binding protein (PvDBP), has been identified as the parasite ligand for Duffy-glycoprotein receptor on human erythrocytes [2,3]. PvDBP gene sequence analysis [4] reveals that it belongs to a family of Plasmodium adhesion proteins found in several Plasmodia including the P. falciparum erythroAbbre6iations: Bv-PvDBPrII, baculovirus recombinant PvDBPrII protein; IL-8, interleukin-8; PvDBPrII, region II of P. 6i6ax Duffy binding protein. Disclaimer: The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. * Corresponding author. Tel.: + 1-301-3199473; fax: + 1301-3199012. E-mail address: [email protected] (C.F. Ockenhouse). 1 Present address: Division of Vaccines and Related Products Applications, OVRR¯CBER¯FDA, Rockville MD, USA.
cyte-binding antigen (EBA-175) and the variant surface antigen PfEMP-1 encoded by 6ar genes [5–7]. In the N-terminal region of PvDBP, a 324 amino acid cysteine-rich stretch or ‘region II’ (PvDBPrII), has been identified as the principal erythrocyte binding domain using transfected COS cells [8,9], making PvDBPrII an attractive vaccine candidate. Substantial quantities of correctly folded PvDBPrII protein are needed to initiate structural and immunological studies. We have expressed PvDBPrII gene in Escherichia coli system; however, the protein is contained predominantly within the insoluble fraction (unpublished observation). The E. coli product did not show appreciable erythrocyte binding activity, similar to a previous report with E. coli-expressed region II-containing construct [10]. In addition, there are no conformation-specific monoclonal antibodies to analyze the structure of refolded PvDBPrII protein from E. coli. Hence, we have focused on the eukaryotic baculovirus system for production
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of PvDBPrII. Here we present the expression, purification and characterization of recombinant PvDBPrII protein produced in the insect cell system. PCR primers, forward: 5%-atgcgcggccgctACGATCTCTAGTGCTATTATA-3% and reverse: 5%atatgaattcTGTCACAACTTCCTGAGTATT-3% (restriction sites shown in lower case), were used to amplify PvDBPrII encoding gene (amino acid residues 198–521; TISSAII…to…NTQEVVT) from genomic DNA of Salvador I strain of P. 6i6ax. PCR product was cloned into the Not I –EcoR I sites of baculovirus transfer plasmid pBSV-8His [11] in-frame with a vector encoded
human complement regulatory factor H-like 1 plasma protein (FHL-1) signal peptide on the amino-terminus (Fig. 1A). The construct also encodes an enterokinase cleavage site followed by a 8× histidine tag at the carboxy-terminus (Fig. 1A). The vector pBSV-8His is designed to direct expressed proteins to a secretory pathway and allow easy purification of the product from culture supernatant of insect cells [12]. The cloned insert was evaluated by DNA sequencing, and recombinant PvDBPrII baculovirus clones were generated essentially as described in the Clontech Laboratories BacPAK6 manual (Palo Alto, CA). Cloned virus stock was amplified from a single plaque.
Fig. 1. Expression and purification of the recombinant Bv-PvDBPrII construct. (A) Schematic view of the PvDBP gene (top) showing the conserved amino acid blocks with other EBPs; shaded areas are N- and C-terminal cysteine-rich regions (N-, C-cys); transmembrane (TM). Expressed Bv-PvDBPrII construct (bottom) shows the extra residues encoded by vector pBSV-8His; (*) indicates translation stop. (B) Coomassie-stained SDS-PAGE analysis of Bv-PvDBPrII during various stages of purification. Lane 1, 6.5× concentrated baculovirus culture supernatant; 2, flow-through of Ni2 + column; 3, pooled fractions containing BvPvDBPrII eluted off Ni2 + column and loaded on SP Sepharose; 4, flow-through of the SP Sepharose column; 5, 1 M NaCl eluate of SP Sepharose containing pure Bv-PvDBPrII protein indicated by the arrow. Molecular weight in kDa indicated on left.
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The 367 amino acid long Bv-PvDBPrII polypeptide has a predicted molecular weight of 41 kDa after FHL-1 signal peptide cleavage. Expressed Bv-PvDBPrII secreted into the culture supernatant migrated close to 40 kDa on SDSPAGE as detected by western blot with anti-6× histidine monoclonal antibody (data not shown). The Bv-PvDBPrII appeared as a compact band on non-reducing gels confirming that the majority is present as a single conformer. Its mobility on SDS-PAGE was decreased upon reduction with DTT confirming its disulphide bonded nature, and the protein did not seem to undergo extensive glycosylation in the baculovirus system as assessed by staining the SDS-PAGE with the GelCode® Glycoprotein Staining Kit (Pierce, Rockford, IL) (data not shown). N-terminus amino acid sequencing of purified Bv-PvDBPrII protein revealed the first six amino acids as Thr – Ile – Ser–Ser–Ala–Ile which corresponds to the first six residues of native PvDBPrII, confirming that the signal peptide was efficiently cleaved during secretion. We found it unnecessary to cleave the 8-histidine tag at the C-terminus, as it had no effect on erythrocyte binding ability of BvPvDBPrII. Recombinant Bv-PvDBPrII was produced in large scale using a Bellco Bioreactor with a 10-l working volume. Sf21 insect cells were grown, in serum-free medium (Cyto-SF9, Kemp Biotechnologies, Frederick, MD), culture was maintained at 27°C and the dissolved oxygen level was at 60% of air saturation. At a density of 1.5× 106 cells ml − 1 the culture was infected with recombinant PvDBPrII baculovirus. Seventy-two hours after infection, supernatant was harvested and concentrated at 4°C using a 10 000 MWCO hollow-fiber ultrafilter (A/G Technology, Woburn, MA) to 1.5 l (a 6.5-fold reduction in volume). The concentrated supernatant was aliquoted and stored at −80°C. Bv-PvDBPrII constituted about 8% of total protein in the 6.5-fold concentrated baculoviral culture supernatant (Fig. 1B, lane 1) as estimated by densitometric analysis of Coomassie stained SDS-PAGE gels. Highly purified fractions of the PvDBPrII were obtained using a two-step purification protocol which was carried out at room temperature. A
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typical purification run was started with 50 ml of concentrated supernatant, it was cleared by centrifugation at 10 000× g for 15 min and 5 ml of 10× PBS (1.5 M NaCl, 17 mM KH2PO4, 50 mM Na2HPO4, pH 7.4) was added to it. The supernatant was passed over a 5-ml Ni-NTA SUPERFLOW column (Qiagen; Valencia, CA), on a FPLC system (Waters, MILLIPORE; Milford, MA). The column was washed extensively with 1× PBS followed by PBS containing 500 mM NaCl and 40 mM imidazole. Bv-PvDBPrII was eluted with a 40 to 200 mM imidazole linear gradient in 50 mM Tris, 0.05% Triton X-100 (pH 7.0). Triton X-100 enhanced the recovery and purity during the second step of purification. The first step of purification on Ni2 + column resulted in up to 63–73% pure protein fractions (Fig. 1B, Lane 3). Elution fractions containing BvPvDBPrII were pooled, diluted fourfold in the same buffer without imidazole, and loaded on a 1-ml SP Sepharose Fast Flow cation exchange column (Amersham–Pharmacia Biotech; Piscataway, NJ). The column was washed with 50 mM Tris buffer containing 100 mM NaCl (pH 7.0) and purified Bv-PvDBPrII was eluted in 50 mM Tris (pH 7.0) containing 1 M NaCl. Eluted protein was dialyzed against 1× PBS overnight and stored at −20°C. Estimated purity of the final product was 92–96% (Fig. 1B, Lane 5). An average of 2 mg Bv-PvDBPrII protein was purified per liter of the baculovirus supernatant. Purified Bv-PvDBPrII protein was analyzed further for its biological activity using an erythrocyte binding assay (see Fig. 2A legend). BvPvDBPrII protein bound Duffy-positive human erythrocytes (Fig. 2A) while no binding was observed with Duffy-negative erythrocytes. Positive binding was also shown with Aotus monkey erythrocytes, but not with Rhesus monkey erythrocytes. This data corroborates the observed binding properties for native PvDBP from P. 6i6ax culture supernatant [2]. In order to evaluate the specificity of this interaction we pretreated human Duffy-positive erythrocytes with either trypsin or chymotrypsin, and assayed these treated erythrocytes for Bv-PvDBPrII-binding activity. Chymotrypsin which is known to remove the Duffy antigen from erythrocytes abolished the
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Fig. 2. Biological and immunological characterization of BvPvDBPrII protein. (A) Erythrocyte binding assays were performed using Aotus, Rhesus, and human erythrocytes (Duffy-positive (pos), Duffy-negative (neg)). Erythrocytes (12 ml of 50% hematocrit) in PBS were added to 500 ml PBS containing 0.1% BSA along with 1 mg Bv-PvDBPrII protein and incubated at 37°C for 30 min. Erythrocytes were washed twice with 0.5 ml PBS and bound protein eluted in 30 ml 1 M NaCl containing 1 mM PMSF. Eluted protein was detected on a western blot immunostained with affinity purified polyclonal anti-Bv-PvDBPrII as the primary antibody. The blot was developed with SuperSignal Chemi-luminescent substrate reagent (Pierce) and exposed on Kodak X-Omat Blue XB-1 film (Eastman Kodak, Rochester, NY). (B) Effect of trypsin or chymotrypsin enzyme treatment on Bv-PvDBPrII binding to human Duffy-positive erythrocytes. (C) Competitive inhibition of Bv-PvDBPrII binding to human Duffy-positive erythrocytes with 0, 0.25 and 2.5 mM IL-8. (D) Recognition of Bv-PvDBPrII by human immune sera in immunoblot (serum dilution 1:1000; top panel). Inhibitory activity of Bv-PvDBPrII binding to Duffy-positive red cells using the same set of sera (bottom panel). (E) Indirect immunofluorescence assay, on methanol fixed Aotus erythrocytes infected with P. 6i6ax Sal I strain, with affinity purified 20 mg ml − 1 rabbit anti-Bv-PvDBPrII antibodies. Fluorescence image of a mature schizont (arrow) containing individual merozoites appearing as dots (magnification × 1000).
binding of Bv-PvDBPrII to Duffy-positive erythrocytes (Fig. 2B). Trypsin treatment which has no effect on the Duffy antigen did not cause any reduction in Bv-PvDBPrII binding to erythrocytes. Recombinant IL-8 has been shown to compete with PvDBPrII for binding to the Duffy antigen on erythrocytes [8,13]. Inhibition of Bv-PvDBPrII binding to human Duffy-positive erythrocytes was done by pre-incubating erythrocytes with two concentrations of recombinant human IL-8 (R&D systems; Minneapolis, MN) for 30 min at 37°C followed by erythrocyte binding assay. IL-8 at 0.25 mM concentration inhibited approximately 75% Bv-PvDBPrII binding (Fig. 2C) and close to 90% inhibition was observed at 2.5 mM estimated by densitometer. In order to assess whether the protein was antigenic, human immune sera collected from adults attending a malaria clinic on the Thai– Burmese border as part of a larger study examining immune responses to P. falciparum and P. 6i6ax antigens (data to be presented elsewhere) were tested by immunoblot for reactivity to recombinant Bv-PvDBPrII. A preliminary examination indicated that PvDBPrII was indeed antigenic, in that the protein reacted to antibodies present in some serum samples (Fig. 2D, lanes 1,2,4,6 top panel). Furthermore, these same serum samples were also analyzed for their ability to block Bv-PvDBPrII binding to human Duffy-positive erythrocytes. Binding inhibition was done by incubating 1 mg Bv-PvDBPrII with 10 ml of human serum (final dilution 1:5) for 1 h at room temperature prior to its addition to the erythrocyte suspension (Fig. 2D, bottom panel). Although some of the sera showed the presence of erythrocyte binding-inhibitory antibodies (lanes 2 and 6, bottom panel), other samples (lanes 1 and 4, bottom panel) had no detectable anti-PvDBP blocking activity despite being highly reactive to PvDBPrII on immunoblot. Further immunologic characterization indicated that Bv-PvDBPrII is immunogenic in addition to being antigenic. Anti-PvDBPrII antibodies affinity-purified from rabbit serum immunized with Bv-PvDBPrII (in Freund’s adjuvant), reacted with merozoites within late stage schizonts on
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indirect immunofluorescence assay (Fig. 2E), similar to the pattern observed previously in P. knowlesi schizonts [14], further establishing the near native structure of the Bv-PvDBPrII. Plasmodium antigens, such as the PvDBPrII, containing a large number of cysteine and aromatic amino acids pose a problem of insolubility during expression in the E. coli system. Although the total yield of PvDBPrII produced in E. coli is considerably larger than baculovirus, the fraction of correctly folded and functional molecules is low as indicated by poor erythrocyte binding characteristics (Dutta et al., unpublished data). The baculovirus system offers several advantages over prokaryotic systems including secretion of the expressed protein into the culture supernatant, correct folding, signal peptide cleavage and posttranslational modifications. Bv-PvDBPrII expressed in baculovirus system showed biological characteristics similar to those reported for native P. 6i6ax PvDBP protein from culture supernatant or COS cell expressed region II. Immunological characteristics of Bv-PvDBPrII confirms the near native structure of recombinant protein. Human immune sera from individuals residing in an endemic area not only recognized Bv-PvDBPrII on western blot and ELISA (data not shown), but in some cases were able to block Bv-PvDBPrII binding to erythrocytes. Interestingly, the presence of high titer antibodies to recombinant PvDBPrII did not necessarily correlate with its ability to block erythrocyte binding in a preliminary analysis of persons exposed to and infected with P. 6i6ax. Further investigation is required to examine the nature of epitopes which elicit inhibitory antibodies and strategies are needed to optimize the production of inhibitory antibodies. Although it is possible that the activity observed in these in vitro erythrocyte binding assays may not reflect all of the possible anti-parasite effects present in vivo, and because of the limitations of in vitro culture of P. 6i6ax, the significance of these results await confirmation in the in vivo primate models of P. 6i6ax. Nevertheless, the baculovirus-expressed Duffy binding protein is an attractive vaccine candidate and plans are underway to immunize
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non-human primates with Bv-PvDBPrII and challenge with P. 6i6ax in the near future.
Acknowledgements We thank the National Research Council (Washington, DC) for funding S. Dutta; Peter F. Zipfel at Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany for the plasmid; Gregory E. Garcia and Deborah R. Moorad at WRAIR for N-terminal sequencing; Connie L. Howard at the Walter Reed blood bank; Christopher W. Kemp at Kemp Biotechnologies for fermentation; Patrick E. Duffy and V. Ann Stewart at WRAIR for P. 6i6ax parasites; David C. Miles at WRAIR for photography.
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