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Merozoite surface protein 2 of Plasmodium reichenowi is a unique mosaic of Plasmodium falciparum allelic forms and species-specific elements1
Molecular and Biochemical Parasitology 92 (1998) 187 – 192
Short communication
Merozoite surface protein 2 of Plasmodium reichenowi is a unique mosaic of Plasmodium falciparum allelic forms and species-specific elements1 Martin A. Dubbeld, Clemens H.M. Kocken, Alan W. Thomas * Biomedical Primate Research Centre, Department of Parasitology, Lange Kleiweg 157, 2288 GJ Rijswijk, Netherlands Received 22 October 1997; received in revised form 1 December 1997; accepted 9 December 1997
Keywords: Plasmodium reichenowi; Plasmodium falciparum; Malaria; Merozoite; MSA2; MSP2
Plasmodium reichenowi is a chimpanzee malaria parasite that is evolutionarily very closely related to and morphologically highly similar to the human parasite Plasmodium falciparum [1,2]. Limited data on protein encoding sequences are available and to date only sexual stage-specific genes [3–6] and the CSP gene [7] of reichenowi have been sequenced. Here we report the first gene sequence for an asexual blood stage protein of P. reichenowi, the merozoite surface protein 2 (PrMSP2 ), homologous to a P. falciparum strain variable blood-stage merozoite surface antigen [8]. This also represents the first report of an MSP2 Abbre6iations: MSP2, merozoite surface protein 2; Pr, Plasmodium reichenowi; Pf, Plasmodium falciparum. * Corresponding author. Tel.: + 31 15 2842 538; fax: + 31 15 2843 986; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the accession number: Y14731.
gene sequence for a parasite species other than P. falciparum. The function of MSP2 is as yet unknown, although it is a candidate component for a P. falciparum vaccine and has undergone phase I clinical testing in preparation for a phase 2 trial [9]. Comparative structural analyses using homologues from different species can be expected to aid in the identification of functionally critical and constrained regions of vaccine molecules. P. falciparum MSP2 (PfMSP2) contains highly polymorphic elements that provide a basis for the distinction of two major allelic families [10–18]. The primary structure can be divided into five distinct regions [13], where regions one and five are highly conserved N-terminal and C-terminal sequences flanking the central variable regions 2, 3 and 4. These central regions define the two allelic families and comprise variable nonrepetitive sequences (regions 2 and 4), that themselves flank tandem repeats of varying copy number, length and sequence (region 3).
0166-6851/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 2 4 4 - 2
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M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
Fig. 1. Nucleotide and predicted amino acid sequence of the MSP2 gene of P. reichenowi. P. reichenowi DNA was isolated by phenol/chloroform extraction on saponin treated blood from an infected chimpanzee. PCR primers (lower case nucleotides) also containing EcoRI restriction sites at the ends were used to amplify genomic P. reichenowi DNA and the resulting 0.9 kb product was cloned into pGem3Zf. A single pool comprising equal amounts of DNA from six separate recombinant clones, all in the same orientation as determined by restriction enzyme analysis, was sequenced using the Sequenase protocol (USB, Cleveland, OH) and vector- and three PfMSP2 -specific sequencing primers [10]. The resulting DNA sequence was unambiguous, suggesting that the uncloned P. reichenowi stock used to infect the chimpanzee is fairly homogeneous. Putative signal sequence and glycosylphosphatidylinositol (GPI) anchoring sequence [9] are underlined and the central repeat units are shown in italics.
The DNA sequence and the deduced amino acid sequence for PrMSP2 is shown in Fig. 1. The open reading frame of PrMSP2 is 991 nt long and
encodes 296 amino acid residues. In the following description, unless otherwise stated, numbering follows that shown in Fig. 1.
M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
Comparison of the P. reichenowi MSP2 amino acid sequence with the two previously described P. falciparum MSP2 allelic forms FC27 and IC1 [10,11] reveals a similar regional organisation. Region 1 (amino acids 1 – 43) is invariant in PfMSP2, however there are three differences between PrMSP2 and PfMSP2 in this region (amino acids 16, 17 and 30). Region 2 (amino acids 44 – 52), in P. falciparum a non-repetitive, essentially dimorphic region that may also contain point mutations, comprises apparently unrelated species-specific sequence in P. reichenowi. Region 3 (amino acids 51– 112) consists of two tandem repeats of nine amino acids (GTSGTGSGA) that are preceded by an imperfect copy of this repeat unit (GTSGTSDA) and followed by three tandem repeats of seven amino acids (GTSASGV), which are themselves followed by two imperfect copies of the heptamer sequence (GTSGAGV and GTSGAGT). This repeat region is similar to the PfMSP2 IC1-type repeats, but has several distinct characteristics. Firstly, IC1-type repeats are always followed by poly-threonine stretches, which are absent from PrMSP2. Secondly, the repeat units in PrMSP2 contain a threonine residue, which is not found in any P. falciparum MSP2 repeat unit sequenced to date. Thirdly, the IC1type repeat sequences are themselves derived from an underlying hexanucleotide repeat unit GGT GCT [12] and rearrangements always involve a complete hexamer [18]. Alignment of the PrMSP2 repeats with this underlying PfMSP2 hexanucleotide repeat (Fig. 2(A)) shows that, although the hexamer units can be discerned despite substitutions at the first nucleotide of a triplet (nine amino acid repeats) or the first or second nucleotide of a triplet (seven amino acid repeats), the complete hexamer repeat structure is not present in PrMSP2, resulting in an uneven number of repeated amino acids. Region 4 (amino acids 113 – 223) is a non-repetitive polymorphic region of PfMSP2. In PrMSP2 it comprises a complex mixture of sequence that is (1) specific to P. reichenowi (amino acids 113 – 123 and 186–193) (2) that shows 61.3% sequence identity with a P. falciparum Colombian isolate [13] of the FC27-type (Fig. 2(B), amino acids 124 – 185) and (3) that shows 65.5% identity with
189
the P. falciparum FCR3 strain [10] of the IC1-type (Fig. 2(B), amino acids 194–222). Thus PrMSP2 region 4 shows sequence homology to both allelic types of PfMSP2, a combination that has not been observed in any of the P. falciparum isolates sequenced to date. Intriguingly this mosaic of the two P. falciparum allelic forms also contains tandem copies of QEPQTADTTNPT (amino acids 175–199). These overlap the end of the Colombian isolate homology, include a short sequence unique to P. reichenowi in between the regions of homology and extend beyond the start of the FCR3 homology (Fig. 2(B)). Region 5 (amino acids 223–296) is conserved in all PfMSP2 sequences reported to date except for amino acids 256 (7G8 variant sequence [9]) and 260 (7G8 and FC27 variant sequence [10]). However, comparatively extensive differences are evident between PrMSP2 and PfMSP2, such that PrMSP2 differs from PfMSP2 by nine (FC27 strain) or ten amino acids (IC1 strain) in this region At the nucleotide level a strong bias towards nonsynonymous substitutions is observed, both in the conserved region 1 and in the conserved region 5. In region 1 only three nonsynonymous substitutions are observed when PrMSP2 is compared with PfMSP2 FC27 or IC1. In region 5, comparison with the PfMSP2 FC27 allele reveals twelve nonsynonymous and three synonymous substitutions. Comparison with PfMSP2 IC1 reveals an additional nonsynonymous substitution, reflecting the single amino acid difference between FC27 and IC1 in this region. This bias towards nonsynonymous substitutions suggests positive selection for these substitutions, either driven by the host immune system, or by adaptation to other host factors. Alignment of the two P. falciparum alleles with PrMSP2 suggests that the two most related sequences are PfMSP2 IC1 and PrMSP2, as deduced from Unweighted Pair Group Method with Arithmetic Mean Analysis [19]. Additional MSP2 sequences from other P. reichenowi strains would reveal whether FC27-type repeat sequences are also present in the P. reichenowi population. The extensive polymorphism of PfMSP2 protein is reflected in the sequence of PrMSP2 when it is compared to the two allelic types of PfMSP2. Unlike the relatively invariant sexual
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Fig. 2. (A) Alignment of two tandem copies of the PfMSP2 underlying hexanucleotide repeat unit (underlined) and predicted amino acid sequence with PrMSP2 units encoding 9-mer and 7-mer repeats. Nucleotides differing from the consensus are shown in bold. Deletions are represented by -. Similarity to the PfMSP2 consensus is evident, except that the repeat units are not complete hexamer units, resulting in an uneven number of repeated amino acids. (B) Alignment of PrMSP2 region 4 (amino acids 113 – 223) and the best matching P. falciparum MSP2 sequences of the FC27 allelic type (Colombian isolate (Col) [13]) and the IC1 allelic type (FCR3 (FC) [10]). The 12-mer repeat is shown in italics.
stage antigens, where P. reichenowi sequences closely resemble the P. falciparum sequences [3 – 6] and the pre-erythrocytic stage CS protein, where slightly more differences are noted [7], the asexual blood stage protein MSP2 shows considerable differences between P. reichenowi and P. falciparum. The central variable regions of PfMSP2 are surface accessible elements that represent the main target for antibody responses, as was shown by reactivity of immune Aotus monkey sera and
human serum with recombinant protein [10] and ELISA analyses of immune population responses [20]. The different primary structure of this region in PrMSP2 suggests that distinct immune pressure in a different host may have selected for this particular sequence. Jones et al. [21] identified three B-cell epitopes in the conserved regions of PfMSP2: amino acids 21–43 in the N-terminal region and amino acids 231–238 and 245–252 in the C-terminal region of PfMSP2. Saul et al. [22] showed that these conserved epitopes, especially
M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
the N-terminal one, could cross-protect mice from lethal P. berghei infections. The highly similar structure of these regions in PrMSP2 (only a single conservative substitution is observed in the N-terminal peptide and the most C-terminal peptide and a single non-conservative substitution at the N-terminus of the second C-terminal peptide), suggests that these epitopes are functionally constrained. Therefore, as suggested by Jones et al. [21], inclusion of the conserved PfMSP2 epitopes in a subunit vaccine might be a valuable addition to a multi-component vaccine that will improve effectivity of the vaccine against all circulating P. falciparum parasites. A recent pilot study in humans on the safety, immunogenicity and efficacy of a recombinant PfMSP2 protein, comprising the whole molecule except for the signal peptide and the GPI anchoring signal [9], showed that the protein was immunogenic, but no protective effect from infection was obvious. Further studies on immunogenicity of this protein and the conserved regions of the protein in combination with promising adjuvants are necessary to evaluate the potential of MSP2 as a malaria blood stage vaccine candidate. This is particularly important in view of the published correlation between reduced malaria morbidity and the presence of antibodies to non-repeat regions of PfMSP2 [23]. It will be interesting as more PfMSP2 sequence becomes available to see whether the unique features of PrMSP2 described here are represented in the naturally occurring PfMSP2 diversity.
Acknowledgements The study was supported by grant CT94-0275 from the INCO-DC programme of European Commission DGXII.
References [1] Coatney GR, Collins WE, McWarren W, Contacos PG. The Primate Malarias, Washington DC, US Government Printing Office, 1971, pp. 309–313. [2] Escalante AA, Ayala FJ. Phylogeny of the malaria genus Plasmodium, derived from rRNA gene sequences. Proc Natl Acad Sci USA 1994;91:11373–7.
191
[3] Lal AA, Goldman IF, Campbell GH. Primary structure of the 25-kilodalton ookinete antigen from Plasmodium reichenowi. Mol Biochem Parasitol 1990;43:143 – 6. [4] Moelans IIMD, Lal AA, Konings RNH, Schoenmakers JGG. Sequence of a 16-kilodalton sexual stage and sporozoite surface antigen of Plasmodium reichenowi and comparison with Pfs16 of Plasmodium falciparum. Mol Biochem Parasitol 1991;50:349 – 50. [5] Lal AA, Goldman IF, Collins WE, Kumar N. Sequence of a 27-kilodalton gamete antigen of Plasmodium reichenowi and comparison with Pfg27 of Plasmodium falciparum. Mol Biochem Parasitol 1993;59:175 – 6. [6] Milek RL, Kocken CH, Kaan AM, Jansen J, Meijers H, Konings RN. Plasmodium reichenowi: deduced amino acid sequence of sexual stage-specific surface antigen Prs48/45 and comparison with its homologue in Plasmodium falciparum. Exp Parasitol 1997;87:150 – 2. [7] Lal AA, Goldman IF. Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionary related to the human malaria parasite Plasmodium falciparum. J Biol Chem 1991;266:6686– 9. [8] Smythe JA, Coppel RL, Brown GV, Ramasamy R, Kemp DJ, Anders RF. Identification of two integral membrane proteins of Plasmodium falciparum. Proc Natl Acad Sci USA 1988;85:5195 – 9. [9] Sturchler D, Berger R, Rudin C, et al. Safety, immunogenicity and pilot efficacy of Plasmodium falciparum sporozoite and asexual blood-stage combination vaccine in Swiss adults. Am J Trop Med Hyg 1995;53:423 – 31. [10] Thomas AW, Carr DA, Carter JM, Lyon JA. Sequence comparison of allelic forms of the Plasmodium falciparum merozoite surface antigen MSA2. Mol Biochem Parasitol 1990;43:211 – 20. [11] Smythe JA, Peterson MG, Coppel RL, Saul AJ, Kemp DJ, Anders RF. Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum. Mol Biochem Parasitol 1990;39:227 – 34. [12] Smythe JA, Coppel RL, Day KP, et al. Structural diversity in the Plasmodium falciparum merozoite surface antigen 2. Proc Natl Acad Sci USA 1991;88:1751 – 5. [13] Snewin VA, Herrera M, Sanches G, Scherf A, Langsley G, Herrera S. Polymorpism of the alleles of the merozoite surface antigens MSA1 and MSA2 in Plasmodium falciparum wild isolates from Colombia. Mol Biochem Parasitol 1991;49:265 – 76. [14] Marshall VM, Coppel RL, Martin RK, Oduola AMJ, Anders RF, Kemp DJ. A Plasmodium falciparum MSA2 gene apparently generated by intragenic recombination between the two allelic families. Mol Biochem Parasitol 1991;45:349 – 52. [15] Marshall VM, Coppel RL, Anders RF, Kemp DJ. Two novel alleles within subfamilies of the merozoite surface antigen 2 of Plasmodium falciparum. Mol Biochem Parasitol 1992;50:181 – 4. [16] Marshall VM, Anthony RL, Bangs MJ, Purnomo, Anders RF, Coppel RL. Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 MSA2 in a geo-
M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
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[17]
[18]
[19] [20]
graphically restricted area of Irian Jaya. Mol Biochem Parasitol 1994; 63:13–21. Felger I, Tavul L, Kabintik S, et al. P. falciparum: extensive polymorphism in merozoite surface antigen 2 alleles in an area with endemic malaria in Papua New Guinea. Exp Parasitol 1994;79:106–16. Felger I, Marshall VM, Reeder JC, Hunt JA, Mgone CS, Beck HP. Sequence diversity and molecular evolution of the merozoite surface antigen 2 of Plasmodium falciparum. J Mol Evol 1997;45:154–60. Nei M. Molecular Evolutionary Genetics, New York, Columbia University Press, 1987;293–298. Taylor RR, Smith DB, Robinson VJ, McBride JS, Riley EM. Human antibody response to Plasmodium falciparum merozoite surface protein 2 is serogroup specific and
.
predominantly of the immunoglobulin G3 subclass. Infect Immun 1995;63:4382 – 8. [21] Jones GL, Edmundson HM, Lord R, Spencer L, Mollard R, Saul AJ. Immunological fine structure of the variable and constant regions of a polymorphic malarial surface antigen from Plasmodium falciparum. Mol Biochem Parasitol 1991;48:1 – 10. [22] Saul A, Lord R, Jones GL, Spencer L. Protective immunization with invariant peptides of the P falciparum antigen MSA2. J Immunol 1992;148:208 – 11. [23] Al-Yaman F, Genton B, Anders RF, et al. Relationship between humoral response to Plasmodium falciparum merozoite surface antigen-2 and malaria morbidity in a highly endemic area of Papua New Guinea. Am J Trop Med Hyg 1994;51:593 – 602.
Short communication
Merozoite surface protein 2 of Plasmodium reichenowi is a unique mosaic of Plasmodium falciparum allelic forms and species-specific elements1 Martin A. Dubbeld, Clemens H.M. Kocken, Alan W. Thomas * Biomedical Primate Research Centre, Department of Parasitology, Lange Kleiweg 157, 2288 GJ Rijswijk, Netherlands Received 22 October 1997; received in revised form 1 December 1997; accepted 9 December 1997
Keywords: Plasmodium reichenowi; Plasmodium falciparum; Malaria; Merozoite; MSA2; MSP2
Plasmodium reichenowi is a chimpanzee malaria parasite that is evolutionarily very closely related to and morphologically highly similar to the human parasite Plasmodium falciparum [1,2]. Limited data on protein encoding sequences are available and to date only sexual stage-specific genes [3–6] and the CSP gene [7] of reichenowi have been sequenced. Here we report the first gene sequence for an asexual blood stage protein of P. reichenowi, the merozoite surface protein 2 (PrMSP2 ), homologous to a P. falciparum strain variable blood-stage merozoite surface antigen [8]. This also represents the first report of an MSP2 Abbre6iations: MSP2, merozoite surface protein 2; Pr, Plasmodium reichenowi; Pf, Plasmodium falciparum. * Corresponding author. Tel.: + 31 15 2842 538; fax: + 31 15 2843 986; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the accession number: Y14731.
gene sequence for a parasite species other than P. falciparum. The function of MSP2 is as yet unknown, although it is a candidate component for a P. falciparum vaccine and has undergone phase I clinical testing in preparation for a phase 2 trial [9]. Comparative structural analyses using homologues from different species can be expected to aid in the identification of functionally critical and constrained regions of vaccine molecules. P. falciparum MSP2 (PfMSP2) contains highly polymorphic elements that provide a basis for the distinction of two major allelic families [10–18]. The primary structure can be divided into five distinct regions [13], where regions one and five are highly conserved N-terminal and C-terminal sequences flanking the central variable regions 2, 3 and 4. These central regions define the two allelic families and comprise variable nonrepetitive sequences (regions 2 and 4), that themselves flank tandem repeats of varying copy number, length and sequence (region 3).
0166-6851/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 6 - 6 8 5 1 ( 9 7 ) 0 0 2 4 4 - 2
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M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
Fig. 1. Nucleotide and predicted amino acid sequence of the MSP2 gene of P. reichenowi. P. reichenowi DNA was isolated by phenol/chloroform extraction on saponin treated blood from an infected chimpanzee. PCR primers (lower case nucleotides) also containing EcoRI restriction sites at the ends were used to amplify genomic P. reichenowi DNA and the resulting 0.9 kb product was cloned into pGem3Zf. A single pool comprising equal amounts of DNA from six separate recombinant clones, all in the same orientation as determined by restriction enzyme analysis, was sequenced using the Sequenase protocol (USB, Cleveland, OH) and vector- and three PfMSP2 -specific sequencing primers [10]. The resulting DNA sequence was unambiguous, suggesting that the uncloned P. reichenowi stock used to infect the chimpanzee is fairly homogeneous. Putative signal sequence and glycosylphosphatidylinositol (GPI) anchoring sequence [9] are underlined and the central repeat units are shown in italics.
The DNA sequence and the deduced amino acid sequence for PrMSP2 is shown in Fig. 1. The open reading frame of PrMSP2 is 991 nt long and
encodes 296 amino acid residues. In the following description, unless otherwise stated, numbering follows that shown in Fig. 1.
M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
Comparison of the P. reichenowi MSP2 amino acid sequence with the two previously described P. falciparum MSP2 allelic forms FC27 and IC1 [10,11] reveals a similar regional organisation. Region 1 (amino acids 1 – 43) is invariant in PfMSP2, however there are three differences between PrMSP2 and PfMSP2 in this region (amino acids 16, 17 and 30). Region 2 (amino acids 44 – 52), in P. falciparum a non-repetitive, essentially dimorphic region that may also contain point mutations, comprises apparently unrelated species-specific sequence in P. reichenowi. Region 3 (amino acids 51– 112) consists of two tandem repeats of nine amino acids (GTSGTGSGA) that are preceded by an imperfect copy of this repeat unit (GTSGTSDA) and followed by three tandem repeats of seven amino acids (GTSASGV), which are themselves followed by two imperfect copies of the heptamer sequence (GTSGAGV and GTSGAGT). This repeat region is similar to the PfMSP2 IC1-type repeats, but has several distinct characteristics. Firstly, IC1-type repeats are always followed by poly-threonine stretches, which are absent from PrMSP2. Secondly, the repeat units in PrMSP2 contain a threonine residue, which is not found in any P. falciparum MSP2 repeat unit sequenced to date. Thirdly, the IC1type repeat sequences are themselves derived from an underlying hexanucleotide repeat unit GGT GCT [12] and rearrangements always involve a complete hexamer [18]. Alignment of the PrMSP2 repeats with this underlying PfMSP2 hexanucleotide repeat (Fig. 2(A)) shows that, although the hexamer units can be discerned despite substitutions at the first nucleotide of a triplet (nine amino acid repeats) or the first or second nucleotide of a triplet (seven amino acid repeats), the complete hexamer repeat structure is not present in PrMSP2, resulting in an uneven number of repeated amino acids. Region 4 (amino acids 113 – 223) is a non-repetitive polymorphic region of PfMSP2. In PrMSP2 it comprises a complex mixture of sequence that is (1) specific to P. reichenowi (amino acids 113 – 123 and 186–193) (2) that shows 61.3% sequence identity with a P. falciparum Colombian isolate [13] of the FC27-type (Fig. 2(B), amino acids 124 – 185) and (3) that shows 65.5% identity with
189
the P. falciparum FCR3 strain [10] of the IC1-type (Fig. 2(B), amino acids 194–222). Thus PrMSP2 region 4 shows sequence homology to both allelic types of PfMSP2, a combination that has not been observed in any of the P. falciparum isolates sequenced to date. Intriguingly this mosaic of the two P. falciparum allelic forms also contains tandem copies of QEPQTADTTNPT (amino acids 175–199). These overlap the end of the Colombian isolate homology, include a short sequence unique to P. reichenowi in between the regions of homology and extend beyond the start of the FCR3 homology (Fig. 2(B)). Region 5 (amino acids 223–296) is conserved in all PfMSP2 sequences reported to date except for amino acids 256 (7G8 variant sequence [9]) and 260 (7G8 and FC27 variant sequence [10]). However, comparatively extensive differences are evident between PrMSP2 and PfMSP2, such that PrMSP2 differs from PfMSP2 by nine (FC27 strain) or ten amino acids (IC1 strain) in this region At the nucleotide level a strong bias towards nonsynonymous substitutions is observed, both in the conserved region 1 and in the conserved region 5. In region 1 only three nonsynonymous substitutions are observed when PrMSP2 is compared with PfMSP2 FC27 or IC1. In region 5, comparison with the PfMSP2 FC27 allele reveals twelve nonsynonymous and three synonymous substitutions. Comparison with PfMSP2 IC1 reveals an additional nonsynonymous substitution, reflecting the single amino acid difference between FC27 and IC1 in this region. This bias towards nonsynonymous substitutions suggests positive selection for these substitutions, either driven by the host immune system, or by adaptation to other host factors. Alignment of the two P. falciparum alleles with PrMSP2 suggests that the two most related sequences are PfMSP2 IC1 and PrMSP2, as deduced from Unweighted Pair Group Method with Arithmetic Mean Analysis [19]. Additional MSP2 sequences from other P. reichenowi strains would reveal whether FC27-type repeat sequences are also present in the P. reichenowi population. The extensive polymorphism of PfMSP2 protein is reflected in the sequence of PrMSP2 when it is compared to the two allelic types of PfMSP2. Unlike the relatively invariant sexual
190
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Fig. 2. (A) Alignment of two tandem copies of the PfMSP2 underlying hexanucleotide repeat unit (underlined) and predicted amino acid sequence with PrMSP2 units encoding 9-mer and 7-mer repeats. Nucleotides differing from the consensus are shown in bold. Deletions are represented by -. Similarity to the PfMSP2 consensus is evident, except that the repeat units are not complete hexamer units, resulting in an uneven number of repeated amino acids. (B) Alignment of PrMSP2 region 4 (amino acids 113 – 223) and the best matching P. falciparum MSP2 sequences of the FC27 allelic type (Colombian isolate (Col) [13]) and the IC1 allelic type (FCR3 (FC) [10]). The 12-mer repeat is shown in italics.
stage antigens, where P. reichenowi sequences closely resemble the P. falciparum sequences [3 – 6] and the pre-erythrocytic stage CS protein, where slightly more differences are noted [7], the asexual blood stage protein MSP2 shows considerable differences between P. reichenowi and P. falciparum. The central variable regions of PfMSP2 are surface accessible elements that represent the main target for antibody responses, as was shown by reactivity of immune Aotus monkey sera and
human serum with recombinant protein [10] and ELISA analyses of immune population responses [20]. The different primary structure of this region in PrMSP2 suggests that distinct immune pressure in a different host may have selected for this particular sequence. Jones et al. [21] identified three B-cell epitopes in the conserved regions of PfMSP2: amino acids 21–43 in the N-terminal region and amino acids 231–238 and 245–252 in the C-terminal region of PfMSP2. Saul et al. [22] showed that these conserved epitopes, especially
M.A. Dubbeld et al. / Molecular and Biochemical Parasitology 92 (1998) 187–192
the N-terminal one, could cross-protect mice from lethal P. berghei infections. The highly similar structure of these regions in PrMSP2 (only a single conservative substitution is observed in the N-terminal peptide and the most C-terminal peptide and a single non-conservative substitution at the N-terminus of the second C-terminal peptide), suggests that these epitopes are functionally constrained. Therefore, as suggested by Jones et al. [21], inclusion of the conserved PfMSP2 epitopes in a subunit vaccine might be a valuable addition to a multi-component vaccine that will improve effectivity of the vaccine against all circulating P. falciparum parasites. A recent pilot study in humans on the safety, immunogenicity and efficacy of a recombinant PfMSP2 protein, comprising the whole molecule except for the signal peptide and the GPI anchoring signal [9], showed that the protein was immunogenic, but no protective effect from infection was obvious. Further studies on immunogenicity of this protein and the conserved regions of the protein in combination with promising adjuvants are necessary to evaluate the potential of MSP2 as a malaria blood stage vaccine candidate. This is particularly important in view of the published correlation between reduced malaria morbidity and the presence of antibodies to non-repeat regions of PfMSP2 [23]. It will be interesting as more PfMSP2 sequence becomes available to see whether the unique features of PrMSP2 described here are represented in the naturally occurring PfMSP2 diversity.
Acknowledgements The study was supported by grant CT94-0275 from the INCO-DC programme of European Commission DGXII.
References [1] Coatney GR, Collins WE, McWarren W, Contacos PG. The Primate Malarias, Washington DC, US Government Printing Office, 1971, pp. 309–313. [2] Escalante AA, Ayala FJ. Phylogeny of the malaria genus Plasmodium, derived from rRNA gene sequences. Proc Natl Acad Sci USA 1994;91:11373–7.
191
[3] Lal AA, Goldman IF, Campbell GH. Primary structure of the 25-kilodalton ookinete antigen from Plasmodium reichenowi. Mol Biochem Parasitol 1990;43:143 – 6. [4] Moelans IIMD, Lal AA, Konings RNH, Schoenmakers JGG. Sequence of a 16-kilodalton sexual stage and sporozoite surface antigen of Plasmodium reichenowi and comparison with Pfs16 of Plasmodium falciparum. Mol Biochem Parasitol 1991;50:349 – 50. [5] Lal AA, Goldman IF, Collins WE, Kumar N. Sequence of a 27-kilodalton gamete antigen of Plasmodium reichenowi and comparison with Pfg27 of Plasmodium falciparum. Mol Biochem Parasitol 1993;59:175 – 6. [6] Milek RL, Kocken CH, Kaan AM, Jansen J, Meijers H, Konings RN. Plasmodium reichenowi: deduced amino acid sequence of sexual stage-specific surface antigen Prs48/45 and comparison with its homologue in Plasmodium falciparum. Exp Parasitol 1997;87:150 – 2. [7] Lal AA, Goldman IF. Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionary related to the human malaria parasite Plasmodium falciparum. J Biol Chem 1991;266:6686– 9. [8] Smythe JA, Coppel RL, Brown GV, Ramasamy R, Kemp DJ, Anders RF. Identification of two integral membrane proteins of Plasmodium falciparum. Proc Natl Acad Sci USA 1988;85:5195 – 9. [9] Sturchler D, Berger R, Rudin C, et al. Safety, immunogenicity and pilot efficacy of Plasmodium falciparum sporozoite and asexual blood-stage combination vaccine in Swiss adults. Am J Trop Med Hyg 1995;53:423 – 31. [10] Thomas AW, Carr DA, Carter JM, Lyon JA. Sequence comparison of allelic forms of the Plasmodium falciparum merozoite surface antigen MSA2. Mol Biochem Parasitol 1990;43:211 – 20. [11] Smythe JA, Peterson MG, Coppel RL, Saul AJ, Kemp DJ, Anders RF. Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum. Mol Biochem Parasitol 1990;39:227 – 34. [12] Smythe JA, Coppel RL, Day KP, et al. Structural diversity in the Plasmodium falciparum merozoite surface antigen 2. Proc Natl Acad Sci USA 1991;88:1751 – 5. [13] Snewin VA, Herrera M, Sanches G, Scherf A, Langsley G, Herrera S. Polymorpism of the alleles of the merozoite surface antigens MSA1 and MSA2 in Plasmodium falciparum wild isolates from Colombia. Mol Biochem Parasitol 1991;49:265 – 76. [14] Marshall VM, Coppel RL, Martin RK, Oduola AMJ, Anders RF, Kemp DJ. A Plasmodium falciparum MSA2 gene apparently generated by intragenic recombination between the two allelic families. Mol Biochem Parasitol 1991;45:349 – 52. [15] Marshall VM, Coppel RL, Anders RF, Kemp DJ. Two novel alleles within subfamilies of the merozoite surface antigen 2 of Plasmodium falciparum. Mol Biochem Parasitol 1992;50:181 – 4. [16] Marshall VM, Anthony RL, Bangs MJ, Purnomo, Anders RF, Coppel RL. Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 MSA2 in a geo-
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[17]
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