Characterization of a sporozoite antigen common to Plasmodium falciparum and Plasmodium berghei

MOLECULAR

Molecular and Biochemical Parasitology 69 (1995) 239-246

&%HEMICAL PARASITOLOGY

Characterization of a sporozoite antigen common to Plasmodium falciparum and Plasmodium berghei Barbara J. Sina a,*, Craig Wright b, Carter T. Atkinson ‘*l, Ripley Ballou b22, Masamichi Aikawa ‘, Michael Hollingdale aY3 a Biomedical Research Institute, Rockuille, MD, USA b Uniuax, Inc., RockLjille, MD, USA ’ Case Western ReserL!e Vniuersiy, Cleveland, OH, USA

Received 17 August 1994; accepted 12 October 1994

Abstract demonstrated that immunization with Plasmodium fulcipurum sporozoites protected mice against sporozoite infection and that this cross-protection was mediated, at least in part, by anti-sporozoite antibody. The experiments presented in this report show that serum and monoclonal antibodies derived from these protected mice identify a novel 42/54-kDa antigen (designated Circumsporozoite Protein 2 or CSP-2) in both P. falciparum and P. berg& sporozoites. Anti-CSP-2 monoclonal antibody blocks invasion of P. falciparum and P. berghei sporozoites into hepatoma cells in vitro and binds the cell surface of sporozoites. Passive transfer of anti-CSP-2 monoclonal antibody protected mice from P. berghei sporozoite infection. Therefore, CSP-2 appears to play a role in the cross-protective immune response observed. Previous

Plasmodium

studies

berg/z&

Keywords: Plasmodium falciparum; Plasmodium berghei; Sporozoite; Malaria immunity; Monoclonal antibody

1. Introduction Irradiated sporozoites and circumsporozoite (CS) proteins are thought to elicit protection that is largely

* Corresponding author. Entomology Department, 1300 Symons Hall. University of Maryland, College Park, MD 20472, USA. ’ Present address: National Wildlife Health Research Center, P.O. Box 218, Hawaii Volcanoes National Park, HI 96718, USA. ‘Present address: Department of Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA. 3 Present address: Office of Vaccines Research and Review, FDA Center for Biologics Evaluation and Research, 1401 Rockville Pike, Rockville, MD 20852-1448, USA. 0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166.6851(94)00198-7

parasite stage and species specific [l--S]. Recent experiments demonstrated that immunization of mice with Plasmodium falciparum sporozoites protected on average 60% of mice from Plasmodium berghei sporozoite infection [6]. Cross-protection appeared to be specific, as P. falciparum sporozoite immunized mice were protected against P. berghei sporozoite infection but remained susceptible to Plasmodium yoeEii sporozoite infection. Passively transferred immunoglobulin derived from P. Jfalcioarum sooro1 I zoite-immunized mice also protected naive mice against challenge with P. berghei sporozoites indieating that cross-protection is mediated, at least in part, by anti-sporozoite antibody.

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The circumsporozoite (CS) protein, which covers the surface of malaria sporozoites, is thought to be the major protective sporozoite antigen in P. berghei [7] and P. fulciparum [S]. The DNA sequence of the P. berghei CS protein gene [9] revealed a 4-aminoacid sequence shared with the P. falciparum CS protein gene [lO,ll] in the central repeat regions. Although a protective IgM monoclonal antibody with dual specificity for P. falciparum and P. berghei CS proteins was isolated from P. falciparum sporozoite-immunized mice [ 121, mice immunized with the recombinant P. falciparum CS protein repeat vaccine candidate, R,, tet 32, remained susceptible to P. berghei sporozoite infection [6]. This suggested that either P. fulciparum sporozoite immunization elicited antibody to a neutralizing P. berghei CS protein repeat epitope which was not mimicked by the CS repeat vaccine immunization, or that a different P. falciparum sporozoite antigen induced the cross-protective antibody response. Experiments are described in this paper in which a novel sporozoite antigen, designated CSP-2, has been identified in both P. falciparum and P. berghei parasites. The ability of anti-CSP-2 monoclonal antibody to block sporozoite infection in vitro and in vivo suggest that CSP-2 plays a role in the previously observed cross-protective immune responses.

2. Materials and methods

2.1. Sporozoites P. berghei and P. falciparum sporozoite tion, isolation, infection and immunization dures were described previously [f&12].

producproce-

2.2. Monoclonal antibodies Monoclonal antibodies were developed from P. falciparum sporozoite immunized BALB/c mice protected against P. berghei sporozoite infection as previously described [12]. In order to detect non-CS protein sporozoite reactive monoclonal antibodies, a multi-step screening protocol was employed. Supematant media from colonies grown in hypoxanthine/ aminopterin/ thymidine selective media were initially screened for antibody production by ELISA (enzyme-linked im-

69 (1995) 239-246

munosorbent assay) using goat anti-mouse IgG, IgA and IgM specific antibodies (Kirkegaard and Perry Labs, Inc., Gaithersburg, MD). Antibody producing hybridomas were tested for reactivity to the P. fulciparum CS protein repeat peptide (PNAP) coupled to bovine serum albumin. Supernatants from non-CS repeat peptide reactive antibody producing hybridomas were rescreened by ELISA and immunoblots containing P, berghei and P. falciparum sporozoite extracts. Four positive hybridomas were identified that produced IgM antibody reactive with the P. berghei and/or P. falciparum 42/54-kDa sporozoite antigen (Mab 4, 63, 84 and 85). The mouse IgG monoclonal antibodies 2AlO (specific for the P. falciparum CS protein repeat sequence [S]) and 3Dll (specific for the P. berghei CS protein repeat sequence [13]) were kindly supplied by Drs. Nussenzweig and Cochrane (New York University, NY). The mouse IgM monoclonal antibodies Mab 36 (which reacts with both the P. berghei and P. falciparum CS protein repeat sequences) and Mab 2-67 (which is specific for Pseudomonas aeruginosa) were described previously [12]. 2.3. Assays using monoclonal antibodies Passive transfer of monoclonal antibodies to naive mice, monoclonal inhibition of sporozoite invasion of hepatoma cells in vitro assays and immunoperoxidase staining of monoclonal antibodies treated P. berghei liver stage parasites in cultured hepatoma cells were performed as previously described [12]. For immunoelectron microscopy, freshly dissected salivary glands containing sporozoites were fixed in 1% paraformaldehyde containing 0.2% glutaraldehyde in 0.1 M phosphate buffer pH 7.3 for 20 min at room temperature. The infected glands were dehydrated with ethanol and embedded in LR Gold Resin. Ultrathin sections were incubated in Mab 63 and goat anti-mouse IgM colloidal gold probe as previously described [15]. Specificity was confirmed by incubating sections with the colloidal gold probe alone. 2.4. Immunoblotting Immunoblots derived from SDS (sodium dodecyl sulfate) polyacrylamide gels of total sporozoite pro-

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3. Results

teins were treated as previously described [12]. For immunoblots containing total P. berghei liver stage parasite proteins, a 1 cm2 glass cover slip of hepatoma G2-A16 cells infected with 2.5 X 10’ P. berghei sporozoites [14] were washed twice with phosphate buffered saline after 16 or 48 h incubations, lysed in 100 ~1 SDS sample buffer and 20 ~1 lysate used per gel lane. Two dimensional SDS polyacrylamide gels of total P. berghei sporozoite protein (run as prescribed by Pharmacia LKB Biotechnology, Piscataway, NJ) were electroblotted and treated sequentially with Mab 36 then Mab 63 in order to co-identify CS protein and CSP-2 on the same immunoblot. A two dimensional-gel P. berghei sporozoite protein immunoblot was initially incubated with Mab 36 which was detected using iodinated anti-mouse immunoglobulin antibody by autoradiography. The same blot was subsequently reincubated in Mab 63 which was also detected by autoradiography after treatment with iodinated anti-mouse immunoglobulin antibody.

A

69 (1995) 239-246

Immunization with sporozoites elicits antibody primarily to the tandemly repeated amino acid sequences of CS proteins [16]. Therefore, anti-P. falciparum sporozoite sera predominantly react with the P. falciparum CS protein corresponding to 58 kDa, 65 kDa, and 67 kDa on immunoblots [8] and anti-P. berghei sporozoite sera predominantly react with the P. berghei CS protein corresponding to 44 kDa, 52 kDa, and 54 kDa [13]. However, when prechallenge sera from P. fafciparum sporozoite-immunized mice which were subsequently protected against P. berghei sporozoite infection were incubated with blots of total P. berghei sporozoite protein, a 42-kDa protein was detected (Fig. lB, Lane 6). A band of 54 kDa was also often observed (data not shown) and these immunoblots displayed varying ratios of 42kDa to 54-kDa band densities compared to the invariant CS protein pattern observed when identical quantities of the same sporozoite preparation was

B 2

3

4

56

.

Fig. 1. (A) Autoradiograph of immunoblot containing: Lane 1, P. falciparum sporozoite (50000) protein; lane 2, uninfected Anopheles stephensi salivary gland protein; lane 3, P. berghei sporozoite (5000) protein. Immunoblot was incubated in anti-P. berghei sporozoite sera followed by iodinated Protein A. (B) Autoradiograph of immunoblots containing: Lane 1, P. berghei sporozoite protein; lane 2, 48 h P. berghei infected hepatoma cell protein; lane 3, 16 h P. berghei infected hepatoma cell protein; lane 4, uninfected hepatoma cell protein; lane 5, protein molecular weight markers; lane 6, same preparation as lane 1. Lane 1 immunoblot was incubated in anti-P. berghei sporozoite serum followed by iodinated Protein A and autoradiographed overnight. Lanes 2-6, immunoblot from the same gel as lane 1 was incubated in anti-P. falciparum sporozoite sera followed by iodinated Protein A and autoradiographed for 3 days.

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repeatedly run side by side on the same SDS gels. Immunoblots showed that the 42-kDa antigen is present in 16 and 48 h liver stage parasites cultured in vitro (Fig. lB, lanes 3 and 2, respectively). Uninfected total hepatoma cell protein did not react with anti-P. falciparum sporozoite serum (Fig. lB, lane 4). Sera from mice immunized with P. berghei sporozoites also recognized a 42-kDa P. falciparum sporozoite protein (Fig. lA, lane 11 quite distinct in molecular weight from the P. falciparum CS protein. The 42/54-kDa antigen was not detected in uninfected mosquito salivary glands (Fig. lA, lane 2), P. berghei or P. falciparum erythrocytic stage parasites (not shown) or in P. yoelii, Plasmodium vivax or Plasmodium gallinaceum sporozoites by immunoblotting (not shown). The lack of cross-reactive 42/54-kDa antigen in P. yoelii sporozoites correlates to the observation that cross-protected mice remained susceptible to P. yoelii sporozoite infection [6]. In order to better characterize the 42/54-kDa antigen and define the role of this apparently novel P. falciparum sporozoite antigen in protection against P. berghei sporozoite infection, 42/54-kDa antigen specific monoclonal antibodies were developed from BALB/c mice immunized with P. falciparum sporozoites which were protected against P. berghei sporozoite infection. Initially, hybridomas were screened to exclude those producing antibodies to the repeat region of the P. falciparum CS protein. Four hybridomas were identified which produced IgM monoclonal antibodies that reacted with the 42/54-kDa antigen. Monoclonal antibodies 63 and 85 reacted with the antigen in both sporozoite species whereas Mab 4 reacted weakly with only the P. berghei antigen and Mab 84 reacted with only the P. falciparum antigen (Fig. 2A). Therefore, the 42/54kDa antigen appears to contain both species-specific

69 (1995) 239-246

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and cross-reactive epitopes. Each monoclonal antibody reacted to variable extents with both the 42-kDa and 54-kDa proteins indicating that they may represent antigenically related forms of the same antigen. The ratio of the density of the 42-kDa to 54-kDa bands varied in different sporozoite preparations used for immunoblots. However, it is not possible in these experiments to determine whether the 42-kDa and 54-kDa proteins are related by proteolytic processing or other post-translational modifications. Mab 63, which recognized both the P. falciparum and P. berghei 42/54-kDa antigens was used for subsequent experiments. Mab 63 lacked recognition of linear peptide epitopes representing both the repeated and non-repeated regions of the CS proteins of P. berghei and P. falciparum by Pepscan analysis ([12]; data not shown). In order to indisputably distinguish the P. berghei 42/54-kDa antigen from P. berghei CS protein, Mab 63 and Mab 36 (an IgM monoclonal antibody which reacts with both the P. berghei and P. falciparum CS protein repeat sequences [12]) were used on immunoblots of total P. berghei sporozoite proteins separated by two dimensional gel electrophoresis. A two dimensional gel immunoblot was initially treated with Mab 36 to localize the CS protein by autoradiography (Fig. 3A) then reincubated in Mab 63 which was also subsequently detected by autoradiography (Fig. 3B). The two proteins which exhibited distinct pIs in the first dimension isoelectric focusing (IEF) gel migrate to approximately 42-44 kDa in the second dimension SDS gel. P. berghei CS protein, detected by Mab 36 migrated in the first dimension IEF gel to an acidic pI of approximately 4.5. P. berghei CS protein was previously determined to migrate with a p1 of 4.7 in two dimensional gels [17]. The additional spot detected by Mab 63 migrated in the first dimension IEF gel with a basic p1 of approximately 9.0. Therefore,

Fig. 2. (A) Autoradiographs of immunoblots containing P. berghei sporozoite proteins (lanes 1, 4, 6, 8) and P. fakiparum sporozoite proteins (lanes 2, 3, 5, 7) after incubation with anti-CSP-2 antibody containing supematants from: lanes 1,2, hybridoma 4; lanes 3,4, hybridoma 63. Lanes 5,6, hybridoma 84. Lanes 7,8, hybridoma 85, followed by treatment with iodinated anti-mouse Ig. (B) Electron micrograph of P. falciparum sporozoites incubated with anti-CSP-2 monoclonal antibody 63 followed by immunogold treatment (20000 X magnification). (C) Immunoperoxidase stained 16 h P. berghei infected hepatoma cells after incubation with anti-CS protein monoclonal antibody 36. (D) Immunoperoxidase stained 16 h P. berghei infected hepatoma cells after incubation with anti-CSP-2 monoclonal antibody 63.

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Table 1 Inhibition

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of sporozoite

invasion (ISI) of hepatoma

cells

Antibody

Concentration

%ISI vs. P. falciparum

%ISI vs. P. berghei

Mab 63

50 pg ml-’ 250 Fg ml-’ 300 pg ml-’ 0.5 pg ml-’

ND ND 55% 91%

76% 88% ND 99.3%

Mab 36

P. fulciparum or P. berghei sporozoites were added to cultures of hepatoma cells in the absence or presence of the indicated concentrations of anti-CSP-2 monoclonal antibody 63. The level of P. berghei or P. falciparum sporozoite invasion was enumerated microscopically after immunoperoxidase staining using anti-CS protein monoclonal antibody 36. Percent invasion was calculated relative to invasion in control cultures that received no antibody. %ISI = A-B/Ax 100 where A is the average number of sporozoites which have invaded hepatoma cells in control cultures without antibody and B is the average number of sporozoites which have invaded hepatoma cells in test cultures containing antibody.

Fig. 3. (A) Autoradiograph of immunoblot of two dimensional gel containing P. berghei sporozoite proteins after incubation with anti-CS protein monoclonal antibody 36 detected with iodinated anti-mouse Ig.(B) Autoradiograph of above immunoblot after reincubation in anti-CSP-2 monoclonal antibody 63 detected with iodinated anti-mouse Ig.

the 42/54-kDa antigen contains a distinct amino acid composition compared to P. berghei CS protein. Mab 63 was employed in a series of experiments to determine the potential role of the 42/54-kDa sporozoite antigen in the cross-protection observed. Immunoelectron microscopy detected Mab 63 binding diffusely on the surface of P. falciparum sporozoites (Fig. 2B) and therefore, the 42/54-kDa antigen was designated CSP-2 (circumsporozoite protein 2). Specific Mab 63 binding was also detected in association with the parasitophorous vacuole surrounding 16 h P. berghei liver stage parasites by immunoperoxidase staining (Fig. 2D) confirming the immunoblot results presented above (Fig. 1B). Mab 63 neutralized the ability of both P. berghei and P. falciparum sporozoites to invade hepatoma cells in vitro (Table 1). Mab 63 appears to block P. berghei sporozoites at lower concentrations than that used for approximately the same %ISI with P. falciparum sporozoites. Mab 63 inhibitory activity was

significantly lower than anti-CS protein monoclonal antibodies [12]. The hepatoma cell invasion inhibitory activity of Mab 63 against live sporozoites of both species confirms the surface location of the 42/54-kDa antigen and suggests a protective role for anti-CSP-2 antibody in the cross-protection observed. To test whether anti-CSP-2 antibody could protect mice against P. berghei sporozoite infection in vivo, Mab 63 was passively transferred to naive mice. Mab 63 protected 80% of the treated mice from P. berghei sporozoite infection whereas 13.3% of mice were not infected after treatment with a similar dose of the non-sporozoite reactive IgM monoclonal antibody 2-67 (Table 2). Another experiment using a Table 2 Passive transfer of anti-CSP-2

monoclonal

antibody

63

Antibody

Dose ( pg)

% Protected

P value

None Mab 2-67 Mab 63 Mab 36

0 667 720 160

6.7% (l/15) 13.3% (2/15) 80% (12 /15) 100% (15/15)

1.000 0.001 0.0001

_

The IgM monoclonal antibodies Mab 2-67 (anti-P. aeruginosn), Mab 63 (anti-CSP-2) and Mab 36 (anti-CS protein) were injected intravenously into mice which were challenged thirty min later with the bite of ten P. berghei sporozoite infected A. stephensi mosquitoes. Erythrocytic parasitemia was assessed on days 5-10 after P. berghei sporozoite challenge. P values were derived by the x2 method.

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fourfold lower dose of Mab 63 did not provide significant protection to naive mice (data not shown). This implies that the protection conferred by passive transfer of polyclonal anti-P. falciparum sporozoite immunoglobulin to P. berghei sporozoite challenge [6] was due, at least in part, to anti-CSP-2 antibody.

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ing liver stage parasite development and therefore, may be a target for a protective cytotoxic T cell dependent immune response. Further experiments are required to explore the immune mechanisms involved in the cross-sporozoite species protection observed in mice in order to evaluate the potential of CSP-2 as a human malaria anti-sporozoite vaccine candidate.

4. Discussion Serum and monoclonal antibodies derived from mice immunized with P. falciparum sporozoites and protected against P. berghei sporozoite infection [6] identify a novel 42/54-kDa antigen designated CSP2 in both P. falciparum and P. berghei sporozoites. P. falciparum CSP-2 is clearly distinct in molecular weight from P. falciparum CS protein. Although P. berghei CSP-2 is similar in molecular weight to P. berghei CS protein, it displays a distinct p1 when analyzed by two-dimensional electrophoresis. CSP-2 specific monoclonal antibodies were found that react with a 42/54kDa antigen in both malaria sporozoite species suggesting that some conservation of protein structure may induce production of crossreactive antibody. Various measures of evolutionary relatedness have grouped P. falciparum with the rodent and avian malarias whereas P. uiuax appears to be more closely related to the simian malarias [l&20]. However, CSP-2 was not found in P. yoelii, P. L&xxx or P. gallinaceum sporozoites. The P. berghei and P. falciparum CS protein sequences share 34% homology, primarily due to their shared repeat sequences. But excluding the conserved repeat region, the P. berghei CS protein sequence was not found to be more closely related to either P. falciparum, P. vivax or Plasmodium knowlesi CS protein sequences [9]. CSP-2 appears to play a role in the cross-protective immune response observed when mice immunized with P. falciparum sporozoites are protected against P. berghei sporozoite infection. A monoclonal antibody which reacted with CSP-2 of both species blocked the invasion of both species of sporozoites into hepatoma cells in vitro and passive transfer of the monoclonal antibody protected naive mice from P. berghei sporozoite infection, implying that cross-protection may be mediated, at least in part, by anti-CSP-2 antibody. CSP-2 is present dur-

Acknowledgements The authors are grateful for the dedicated technical assistance provided by Angela Appiah, Victoria Harrod, Stephen Lee and Marc Hoffman. This work was supported by U.S.A.I.D. contract DPE 0453-600-4027-00 to M.H. and N.I.H. grant R29AI3048304 to B.J.S.

References 111Nussenzweig,

R.S., Vanderberg, J.P., Sanabria, Y. and Most, H. (1972) Plasmodium berghei: Accelerated clearance of sporozoites from blood as part of the immune mechanism in mice. Exp. Parasitol. 31, 88-97. El Nardin, E.H. and Nussenzweig, R.S. (1978) Stage specific antigens on the surface membrane of sporozoites of malaria parasites. Nature 274, 55-57. 131 Clyde, D.F., McCarthy. V.C., Miller, R.M. and Woodward, W.E. (1975) Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. Am. J. Trop. Med. Hyg. 24. 397-401. R.W., Cochrane. A.H., Nussenzweig, V. and 141 Gwadz, Nussenzweig, R.S. (1979) Preliminary studies on vaccination of Rhesus monkeys with irradiated sporozoites of Plasmodium knowlesi and characterization of surface antigens of these parasites. Bull. WHO 57 Suppl. 1. 165-173 [51Nussenzweig, R.S., Vanderberg, J.P., Most, H. and Or-ton, C. (1969) Specificity of protective immunity produced by Xirradiated Plasmodium berghei sporozoites. Nature 222, 488-489. [61Sina, B.J., Rosario, V.E., Woollett, G., Sakhuja, K. and Hollingdale, M.R. (1993) Plasmodium falciparum sporozoite immunization protects against Plasmodium berghei sporozoite infection. Exp. Parasitol. 77, 129-135. [71Potocnjak, P., Yoshida, N., Nussenzweig, R.S. and Nussenzweig, V. (1980) Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. J. Exp. Med. 151, 1504-1513 k31Nardin, E.H., Nussenzweig, V.. Nussenzweig, R.S.. Collins, W.E., Harinasuta, K.T., Tapchaisri, P. and Chomcham, Y. (1982) Circumsporozoite proteins of human malaria parasites

246

[9]

[lo]

[ll]

[12]

[13]

[14]

B.J. Sina et al. /Molecular

and Biochemical Parasitology

Plasmodium falciparum and Plasmodium viuax. J. Exp. Med. 156, 20-30. Weber, J.L., Egan, J.E., Lyon, J.A., Wirtz, R.A., Charoenvit, Y., Maloy, W.L. and Hockmeyer, W.T. (1987) Plasmodium berghei: Cloning of the circumsporozoite protein gene. Exp. Parasitol. 63, 295-300. Dame, J.B., Williams, J.L., McCutchan, T.F., Weber, J.L., Wirtz, R.A., Hockmeyer, W.T., Maloy, W.L., Hayes, J.D., Schneider, I., Roberts, D., Saunders, G.S., Reddy, E.P., Diggs, C.L. and Miller, C.L. (1984) Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite, Plasmodium falciparum. Science 225,593-599. Enea, V., Ellis, J., Zavala, F., Arnot, D.E., Asavanich, A., Masuda, A., Quakyi, I. and Nussenzweig, R.S. (1984) DNA cloning of Plasmodium falciparum circumsporozoite gene: Amino acid sequence of the repetitive epitope. Science 225, 628-630. Sina, B.J., Wright, C., Ballou, R. and Hollingdale, M. (19921 A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Exp. Parasitol. 74, 431-440 Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Nussenzweig, V. and Aikawa, M. (1980) Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207, 71-73. Hollingdale, M.R., Leland, P., Leef, J.L. and Beaudoin, R.L.

[15]

[16]

[17]

[18]

[19]

[20]

69 (1995) 239-246

(1983) In vitro cultivation of exoerythrocytic stage of Plasmodium berghei in a hepatoma cell line. Am. J. Trop. Med. Hyg. 32, 683-690 Aley, S.B., Atkinson, CT., Aikawa, M., Maloy, W.L. and Hollingdale, M.R. (1986) Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells. J. Parasit. 73, 1241-1247 Zavala, F., Cochrane, A.H., Nardin, E.H., Nussenzweig, R.S. and Nussenzweig, V. (1983) Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J. Exp. Med. 157, 1947-1957. Yoshida, N., Potocnjak, P., Nussenzweig, V. and Nussenzweig, R.S. (1981) Biosynthesis of Pb44, the protective antigen of sporozoites of Plasmodium berghei. J. Exp. Med. 154, 1225-1236. McCutchan, T.F., Dame, J.B., Miller, L.H. and Barnwell, J. (1984) Evolutionary relatedness of Plasmodium species as determined by the structure of DNA. Science 225, 808-811. Waters, A.P., Higgins, D.G. and McCutchan, T.F. (1991) Plasmodium falciparum appears to have arisen as a result of lateral transfer between avian and human hosts. Proc. Natl. Acad. Sci. USA 88, 3140-3144. Waters, A.P., Higgins, D.G. and McCutchan, T.F. (1993) The phylogeny of malaria: A useful study. Parasit. Today 9, 246-250.