Monoclonal antibodies (MAbs) have been produced against Plasmodium ovale sporozoites and used to characterize the circumsporozoite (CS) protein.
Vol. 56, No. 2
INFECTION AND IMMUNITY, Feb. 1988, p. 376-379
0019-9567/88/020376-04$02.00/0
Circumsporozoite Protein of the Human Malaria Parasite Plasmodium ovale Identified with Monoclonal Antibodies PATRICIA M. PROCELL,* WILLIAM E. COLLINS, AND GARY H. CAMPBELL Malaria Branch, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control,
Atlanta, Georgia 30333 Received 24 September 1987/Accepted 11 November 1987
Monoclonal antibodies (MAbs) have been produced against Plasmodium ovale sporozoites and used to characterize the circumsporozoite (CS) protein. Six MAbs were produced, and all were species specific. By using Western blot (immunoblot) analysis, three polypeptides were detected: a predominant 51,000-Mr polypeptide and two presumed precursor 57,000- and 67,000-M, molecules. The presence of a repeating epitope in the CS protein of P. ovale was demonstrated by using one of the MAbs in a single-antibody two-site enzyme immunoassay. Three MAbs recognized epitopes on the surfaces of sporozoites; the presence of at least one other epitope within the CS protein, but not on the surfaces of P. ovale sporozoites, was also demonstrated.
The circumsporozoite (CS) proteins of malaria parasites are the major proteins on the surfaces of mature salivary gland sporozoites. Early studies demonstrated two intracellular precursor forms of the CS protein which have higher molecular weights and isoelectric points. The resultant membrane-bound CS protein is thought to be formed by the sequential removal of two small, basic polypeptides from the higher-molecular-weight precursor molecule (16). Biochemically, CS proteins are characterized by a region of tandemly repeated amino acid sequences. These sequences vary both in the number of amino acid residues (from 4 to 11 amino acids per repeat) and in the number of tandem repeats per molecule for the different species studied to date (10). Monoclonal antibodies (MAbs) have been produced against sporozoites of several Plasmodium species, including the human malaria parasites, Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae (5, 9), and have been used to characterize the respective CS proteins of those parasites. All of the MAbs produced to date by immunization with Plasmodium sporozoites are directed against CS proteins (17). In addition, MAbs produced against several Plasmodium species have been shown to abolish the infectivity of sporozoites in vivo when the MAbs were incubated with the sporozoites before inoculation (4, 8, 15). MAb-based immunoassays have been developed with P. falciparum, P. vivax, and P. malariae CS protein-specific MAbs to detect infected mosquitoes (1, 14, 18; F. H. Collins, P. M. Procell, W. E. Collins, and G. H. Campbell, Am. J. Trop. Med. Hyg., in press). However, the CS protein of Plasmodium ovale has not been characterized previously, as a result of the limited availability of sporozoites. This paper describes the production of MAbs generated against P. ovale sporozoites and their use in characterization of the CS protein of P. ovale.
P. vivax and of the Uganda I/CDC strain of P. malariae were also obtained from mosquitoes fed infected chimpanzee blood through membranes. P. falciparum (Honduras I/CDC strain, B3 clone) sporozoites were obtained by feeding mosquitoes gametocytes produced by in vitro culture. Sporozoites were collected in a similar manner from mosquitoes infected by feeding rhesus blood infected with Plasmodium cynomolgi (Gombak, PT-1, PT-2, or Smithsonian strains or P. cynomolgi bastianellii), P. inui (N-34, Leucosphyrus, OS, or Celebes I strains), Plasmodium simiovale, or Plasmodium brasilianum (Peruvian I strain). Monoclonal antibodies. All mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Female BALB/ cByJ mice were immunized intravenously with 104 fresh salivary gland sporozoites three times at 1-week intervals. The immune spleen cells were fused with the non-immunoglobulin-secreting tumor cell line SP2/0 3 days after the last immunization (11). After 14 days, supernatants were screened for P. ovale-specific antibody by an indirect fluorescent antibody (IFA) assay, and antibody-secreting cell lines were cloned by limiting dilution. The resultant cloned lines were maintained in the logarithmic phase of growth in Opti-MEM (GIBCO Laboratories, Grand Island, N.Y.) with 2 to 4% fetal bovine serum. MAb-containing ascitic fluids were produced in hybridoma-bearing (BALB/cJ x C57BL/6J)F1 mice. MAb was purified from ascitic fluids or culture supernatants with Sephadex-200 (Pharmacia Fine Chemicals, Piscataway, N.J.) column chromatography. IFA assay and CSP reaction. Sporozoites from infected salivary glands were dissected into normal saline with 20% fetal bovine serum for preparation of slides. The sporozoites were allowed to air dry or were glutaraldehyde fixed before air drying. The anti-sporozoite MAbs were detected with fluorescein isothiocyanate-labeled, goat anti-mouse immunoglobulin G (IgG) (heavy- and light-chain specific; Tago, Burlingame, Calif.). Subclass determinations were made with subclass-specific fluorescein isothiocyanate-labeled anti-mouse reagents (Litton Bionetics, Rockville, Md.). The circumsporozoite protein precipitin (CSP) reaction was performed by dissecting infected salivary glands directly into antibody-containing ascitic fluid (13). Polyacrylamide gel electrophoresis and Western blot (immunoblot). Sporozoites dissected into saline were diluted and lysed with an equal volume of sample buffer (0.06 M Tris
MATERIALS AND METHODS Sporozoites. P. ovale sporozoites were obtained by feeding Anopheles stephensi and Anopheles gambiae mosquitoes chimpanzee blood infected with the Nigerian I/CDC strain of P. ovale through membranes (2). Sporozoites were dissected into normal saline (0.85% NaCl) and used immediately or stored frozen at -70°C. Sporozoites of the Chesson strain of *
Corresponding author. 376
CS PROTEIN OF P. OVALE
VOL. 56, 1988 TABLE 1. Characteristics of P. ovale sporozoite MAbsa MAb
Isotype
CSP/IFAb
in Competition ELISAC two-site
IgG3
+ + +
110-54.3 110-92.6 110-138.6
IgM IgM
+/+ +/+ ND/+
110-90.11 110-143.14 110-156.3
IgG2a IgGl IgGl
ND/ND/ND/-
a All MAbs, except for 110-156.3, identified the 51,000-Mr protein by Western blot analysis. b CSP reaction/IFA reactivity with glutaraldehyde-fixed sporozoites. ND, Not done. c Ability (+) or inability (-) to compete with HRP-labeled MAb 110-54.3 in a two-site ELISA.
hydrochloride, 5% sodium dodecyl sulfate, 0.7 M 2-mercaptoethanol), heated for 10 min at 65°C, and electrophoresed through a 3.3 to 22% polyacrylamide gradient gel (12); 105 sporozoites were used for each gel. The proteins, electrophoretically separated under reducing conditions, were blotted onto nitrocellulose at 100 V for 2 h with a Trans Blot apparatus (Bio-Rad Laboratories, Richmond, Calif.). The nitrocellulose strips were reacted with MAbs diluted in phosphate-buffered saline-0.3% Tween 20. Reaction of MAbs with specific polypeptides was determined with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (BioRad) with 3,3'diaminobenzidene and H202 as the enzyme substrate. Molecular weight determinations were made with prestained molecular weight markers (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) as standards. Two-site ELISA. A single-antibody two-site enzymelinked immunosorbent assay (ELISA) was used to determine the presence of a repeating epitope within the CS protein of P. ovale (16). Affinity-purified MAb (110-54.3) (2 jig/ml) was incubated in Immulon II (Dynatech Laboratories, Inc., Alexandria, Va.) microdilution plates overnight at room temperature; the unbound sites were then blocked with phosphate-buffered saline containing 0.5% casein and 0.1% Tween 20. Sporozoites, solubilized for 1 h in 0.0625% Nonidet P-40, were added and incubated for 2 h and then detected with 2 jig of the same MAb per ml, which was HRP labeled (commercially prepared by Kirkegaard and Perry Laboratories, Gaithersburg, Md.) per ml. Specific binding was determined by subtracting the absorbance units (A414) of wells without sporozoites from the A414 of wells with sporozoites. Competitive binding ELISA. A single-antibody two-site ELISA was used with MAb 110-54.3 bound to the solid phase. Unlabeled MAbs (1 to 20 jig/ml) were preincubated with 1,000 Nonidet P-40-solubilized P. ovale sporozoites for 1 h at room temperature, and then the mixture was added to the solid phase and incubated for an additional hour. Any sporozoite proteins bo-und to the solid-phase MAb were detected with HRP-labeled MAb 110-54.3. The percent reduction of binding was calculated by dividing the difference of the absorbance of control wells (anti-P. vivax sporozoite MAb) and test wells (test MAb) by absorbance of the control wells and then multiplying by 100 as follows: % reduction = [(A414 of control wells - A414 of test wells)/ (A414 of control wells)] x 100. RESULTS Properties of the MAbs. The fusion of cells from a single immune mouse spleen resulted in six MAb-secreting cloned
377
hybridoma cell lines. All antibody-secreting cell lines were initially selected with air-dried sporozoites in the IFA assay. The isotypes of the MAbs were determined: two were IgM, two were IgGl, one was IgG2a, and one was IgG3 (Table 1). To determine if the MAbs reacted with surface determinants on the sporozoites, the MAbs were incubated with glutaraldehyde-fixed sporozoites (Table 1). Three MAbs reacted with surface sporozoite epitopes (110-54.3, 110-92.6, and 110-138.6), but the remaining MAbs, which did react with air-dried sporozoites, did not react with the glutaraldehydefixed sporozoites (110-143.14, 110-90.11, and 110-156.3). CSP reactions were performed with freshly dissected P. ovale sporozoites and two MAbs that reacted with glutaraldehyde-fixed sporozoites. Both of these MAbs (110-54.3 and 110-92.6) were able to mediate a positive CSP reaction (Fig. 1). Species cross-reactivities of the MAbs were determined by using the IFA assay and air-dried sporozoites of various human and simian malaria parasites. No cross-reactivities were seen between the MAbs and sporozoites of the human malaria parasites, P. falciparum, P. vivax, and P. malariae, and the following simian parasite species: P. cynomolgi (P. cynomolgi bastianellii and Smithsonian, Gombak, PT-1, and PT-2 strains), Plasmodium inui (N-34, Leucosphyrus, Celebes I, and OS strains), P. simiovale, and P. brasilianum (Peruvian I strain). Characterization of the CS protein. The polypeptides recognized by the MAbs were identified by polyacrylamide gel electrophoresis and Western blot analysis. Five of six MAbs detected a 51,000-Mr predominant band (Fig. 2). Two fainter 57,000- and 67,000-Mr polypeptides were also identified and were most easily seen with MAb 110-54.3 (lane 2). Western blot analysis showed that all but one of the antibodies recognized the same predominant polypeptide. One antibody, 110-156.3 (lane 7), which was reactive in the IFA assay with air-dried sporozoites, did not react with any polypeptide. A control MAb specific for P. vivax sporozoites did not detect any polypeptides (lane 1). A single-antibody two-site ELISA was used to demonstrate the presence of a repeating epitope in the CS protein of P. ovale. One MAb, 110-54.3, was successfully used to capture and detect the solubilized sporozoite proteins. Only CS protein from a P. ovale sporozoite extract could be detected in this assay (Fig. 3); there was no reaction with P.
~ ~ exI . W
B
FIG. 1 .(A) MAb 110-92.6 producing
a
positive CSP reaction
when P. ovale sporozoites were incubated with MAb-containing ascitic fluid (B) An anti-P. vivax MAb incubated with P. ovale
sporozoites, demonstrating no precipitate.
378
PROCELL ET AL.
INFECT. IMMUN. 100
2
1
3
4
5 6
7
Mol.Wt. 80
-97K
67K-57K -51 K-p
z 0
M60 z Iz Z40
-68K
_
.-
w
-43K
a.
20
-25.7K FIG. 2. Western blot analysis of P. ovale sporozoite polypeptides. MAbs produced against P. ovale sporozoites were incubated with an unrelated control MAb (an anti-P. vivax sporozoite MAb) (lane 1), anti-P. ovale MAbs (lanes 2 to 7), 110-54.3 (lane 2), 110-92.6 (lane 3), 110-90.11 (lane 4), 110-138.6 (lane 5), 110-143.14 (lane 6), or 110-156.3 (lane 7). The 67,000-, 57,000-, and 51,000-M, proteins are indicated by the arrows labeled 67K, 57K, and 51K, respectively. Known molecular weight (Mol.Wt.) markers are shown to the right.
vivax, P. falciparum, or P. malariae sporozoite proteins. Recognition of different epitopes on the CS protein by anti-P. ovale MAbs. Three MAbs reacted with surface epitopes on sporozoites, and three did not; yet all but one of the MAbs reacted with the same polypeptides as determined by Western blotting. Competitive inhibition experiments were performed to determine whether any of the MAbs recognized identical epitopes on the CS protein. A single-antibody two-site ELISA was used. The homologous MAb, 110-54.3, inhibited binding of itself as expected (Fig. 4). In addition, two other MAbs, 110-138.6 and 110-92.6, inhibited binding of 110-54.3, although the homologous MAb inhibited at lower concentrations than the other two antibodies, suggesting that these three MAbs recognized the same or related epitopes within the CS protein.
-9 C
0.6
C
0.4
0
0.2
CO
m
a
40
a
O o
P. ovale
m
m
=
m
CD
P. malariae
P.
m
=
CD In =4
falciparum
I
m
m
m
° 3n o
P. vivax
SPOROZOITES FIG. 3. Single-antibody two-site ELISA for detecting repeating epitopes within the CS protein using anti-P. ovale MAb 110-54.3. Sporozoites were solubilized with 0.0625% Nonidet P-40 and diluted to contain the appropriate amount of sporozoites in 50 ,ul of buffer. O.D., Optical density.
0
5
10
15
20
UG/ML UNLABELED MAB ADDED
FIG. 4. Competitive inhibition of binding of anti-P. ovale MAbs for solubilized sporozoite proteins with MAb 110-54.3. MAb 11054.3 was bound to the solid phase. Unlabeled MAbs were preincubated with 1,000 P. ovale sporozoites and then added to the solid phase. Percent inhibition of binding of HRP-labeled MAb 110-54.3 was determined. Symbols: 0, 110-54.3; A, 110-92.6; *, 110-138.6; 0, 110-143.14; A, 110-90.11; O, 110-156.3. MAB, MAb.
DISCUSSION Monoclonal antibodies were generated against P. ovale sporozoites and used to characterize the CS protein of P. ovale (Table 1). Three MAbs recognized epitopes present on the surfaces of sporozoites. Two of these MAbs, 110-92.6 and 110-138.6, were shown to competitively inhibit binding of the other surface-reactive MAb, 110-54.3, in the singleantibody two-site ELISA. Currently, without synthetic peptides to determine the fine specificities of the MAbs, we cannot be certain whether the three surface-reactive MAbs recognize the same epitope within the CS protein or rather, structurally adjacent epitopes. In addition to epitopes of the CS protein exposed on the surface of the sporozoite, at least one other epitope within the CS protein was identified by the other MAbs described in this study. Three antibodies (110143-14, 110-90.11, and 110-156.3) did not react with surface determinants; however, two of these did react with the 51,000-Mr CS protein. These antibodies may recognize epitopes buried in the membrane of the sporozoite or otherwise unexposed in the glutaraldehyde-fixed sporozoites used in the IFA assay. Five of the six MAbs recognized a predominant 51,000-Mr polypeptide and to a lesser degree, the two 57,000- and 67,000-Mr polypeptides. These two higher-molecular-weight polypeptides are presumed precursor molecules of the membrane-bound CS protein; however, the relationship between the presumed precursor molecules and the predominant surface-bound protein of sporozoites remains obscure. One MAb reacted with air-dried sporozoites but did not react with glutaraldehyde-fixed sporozoites and did not identify any sporozoite polypeptides on the Western blot, suggesting that this MAb may react with a protein unrelated to the CS protein. Perhaps the epitope recognized by this MAb was destroyed during electrophoresis or Western blotting, resulting in an altered configuration of the epitope. Alternatively, possible low affinity of the MAb could account for the lack of reactivity with any sporozoite proteins on the Western blot. One of the P. ovale MAbs that mediated the CSP reaction (110-54.3) was used in a single-antibody two-site assay to demonstrate the presence of at least two identical epitopes
CS PROTEIN OF P. OVALE
VOL. 56, 1988
within the CS protein. Since only one MAb was used to make this determination, the presence of only one repeating epitope was confirmed. Ultimately, genomic analysis and amino acid sequencing must be used to confirm the presence of repeating subunits, to determine the length and number of the repeat(s), and to determine the possible presence of different repeating subunits. Early studies suggested that CS proteins were species specific. More recently, several exceptions have been noted. Anti-P. malariae MAbs cross-react with P. brasilianum sporozoites (5), and some anti-P. vivax MAbs cross-react with some strains of P. cynomolgi sporozoites (6, 7). However, no cross-reactions between sporozoites of the human malaria parasites have yet been demonstrated; similarly, the anti-P. ovale MAbs do not cross-react with any other human malaria sporozoites. In addition, several strains of simian species were tested and no cross-reactions were found, suggesting that antigenically, and possibly evolutionarily, P. ovale is not closely related to any of the simian species of malaria parasites examined, including P. simiovale, a morphologically and biologically related simian parasite (3). Species specificity is an important prerequisite to the use of MAbs in entomologic and transmission studies. All MAbbased immunoassays to detect Plasmodium-infected mosquitoes have used a single antibody two-site assay (1, 14, 18; Collins et al., in press), capitalizing on the presence of repeated epitopes within CS proteins. Studies are presently under way to use one of the MAbs described in this study (110-54.3) in an ELISA to detect P. ovale-infected mosquitoes in Africa and Asia. ACKNOWLEDGMENTS We thank Frank H. Collins for helpful discussions, critical review of the manuscript, and provision of P. ovale sporozoites. This work was supported by PASA number BST-0453-P-HC2086-03 from the U.S. Agency for International Development and Public Health Service grant RR-00165 from the National Institutes of Health, Division of Research Resources, to the Yerkes Regional Primate Center, Emory University, Atlanta, Ga. LITERATURE CITED 1. Burkot, T. R., F. Zavala, R. W. Gwadz, F. H. Coflins, R. S. Nussenzweig, and D. R. Roberts. 1984. Identification of malariainfected mosquitoes by a two-site enzyme-linked immunosorbent assay. Am. J. Trop. Med. Hyg. 33:227-231. 2. Chin, W., and P. G. Contacos. 1966. A recently isolated African strain of Plasmodium ovale. Am. J. Trop. Med. Hyg. 15:1-2. 3. Coatney, G. R., W. E. Collins, M. Warren, and P. G. Contacos. 1971. The primate malarias. U.S. Department of Health, Education, and Welfare, Washington, D.C. 4. Cochrane, A. H., J. W. Barnweli, W. E. Collins, and R. S. Nussenzweig. 1985. Monoclonal antibodies produced against
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