Gene polymorphism of Plasmodium falciparum merozoite surface proteins 4 and 5

Molecular & Biochemical Parasitology 142 (2005) 110–115

Short communication

Gene polymorphism of Plasmodium falciparum merozoite surface proteins 4 and 5夽 Hannah E.J. Polson a,∗ , David J. Conway b , Thierry Fandeur c , Odile Mercereau-Puijalon d , Shirley Longacre a a

Laboratoire de Vaccinologie Parasitaire, CNRS URA 2581, Institute Pasteur, Paris, France b The London School of Hygiene and Tropical Medicine, London, UK c Laboratoire d’Epid´ emiologie Mol´eculaire, Institut Pasteur du Cambodge, Phnom Penh, Cambodia d Unit´ e d’Immunologie Mol´ecularie des Parasites, CNRS URA 2581, Institut Pasteur, Paris, France Received 23 December 2004; accepted 13 February 2005

Keywords: Polymorphism; MSP4; MSP5; Plasmodium falciparum

Malaria is responsible for approximately 2 million deaths per year worldwide, mostly African children under 5 years old, and places an enormous public health burden on many of the world’s poorest countries. This burden is increasing at an alarming rate, as drug resistance in both the parasite and its mosquito vectors spreads, exacerbating the urgent need for an effective vaccine. The most promising blood stage vaccine candidates examined so far are merozoite surface proteins (MSPs), including MSP1 and MSP2, and an apical membrane antigen (AMA1) [1–3]. However, many surface antigen genes display a disproportionately high proportion of non-synonymous single nucleotide polymorphisms (nsSNPs) compared to genes coding for proteins that are not accessible to immune effectors [4–6] and some of these nsSNPs encode radical amino acid substitutions that may be clustered within the regions of the peptide most accessible to the host immune system [7]. Such amino acid polymorphisms could function in immune evasion by altering both B and T cell epitopes [4,8]. However, a large proportion of gene polymorphisms can be selectively neutral, Abbreviations: MSP, merozoite surface protein; nsSNP, nonsynonymous single nucleotide polymorphism; sSNP, synonymous single nucleotide polymorphism; EGF, epidermal growth factor; GPI, glycosylphosphatidylinositol; PNG, Papua New Guinea 夽 Note: Nucleotide sequence data reported in this paper are available via GenBank, under Accession Nos. AY861554–AY861651. ∗ Corresponding author at. Laboratoire de Vaccinologie Parasitaire, CNRS URA 2581, Institut Pasteur 25 Rue Du Dr Roux, 75724 Paris, France. Tel.: +33 1 40 61 35 49; fax: +33 1 45 68 84 32. E-mail address: [email protected] (H.E.J. Polson). 0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2005.02.009

so studies are required to test their immunological relevance [9,10]. Two recently identified merozoite surface antigens, Plasmodium falciparum merozoite surface proteins 4 and 5 (MSP4 and MSP5), are being considered as constituents of a potential multi-component vaccine. The msp4 and msp5 genes both code for 272 residue proteins, each with a single Cterminal EGF-like domain and GPI attachment motif [11,12] and are located in tandem on chromosome 2, just upstream of msp2. Membrane association at the merozoite surface has been demonstrated for both proteins, and human immune sera have been shown to react with recombinant MSP4 expressed in Escherichia coli [13,14]. In three murine species of Plasmodium, P. yoelii, P. chabaudi and P. berghei, there is only a single gene at the MSP4 and MSP5 locus, which shows some degree of homology to each [15–17]. This gene is denoted MSP4/5 and has been used to investigate protective immunity in the P. yoelii lethal challenge model [18]. MSP4/5 has been shown to confer protection using a variety of immunisation strategies, and efficacy is maximised when delivered in conjunction with MSP119 [18–21]. In addition, there appears to be no strain specificity in immune responses induced by the murine MSP4/5 protein [22]. In P. falciparum, msp4 and msp5 each have a single intron at homologous locations [23]. Although a cluster of polymorphic sites is found near the N-terminus of MSP4, no significant departure from a pattern of neutral evolution has been detected [24–26]. Surprisingly, for a Plasmodium surface antigen, the msp5 gene was found to be nearly identical in several laboratory parasite strains [12] and in isolates from Vietnam [25]. Transcripts of both

H.E.J. Polson et al. / Molecular & Biochemical Parasitology 142 (2005) 110–115

genes have been detected in sporozoites and hepatic stage parasites [27,28]. Non-blood-stage protein expression has been confirmed for MSP4, which at the time of identification was named sporozoite and liver stage antigen (SALSA) [29,30]. Immunological studies of this 83 amino acid fragment of MSP4 have revealed the presence of both sporozoite and hepatic stage immune effecter epitopes [31,32] and a recent study has shown that two regions of the SALSA sequence (MSP4 residues 76–92 and 113–135) can specifically bind to hepatic cells [33]. To expand the data sets available for analysis of MSP4 and MSP5, we undertook to analyse gene sequences from parasite isolates collected in several different locations. Two villages in Senegal, Ndiop (n = 27) and Dielmo (n = 14), with seasonal and perennial transmission, respectively [34,35], the Battambang province of Cambodia (n = 12) and Sri Lanka (n = 4), where transmission is less intense. The presence of multiple infections was evaluated for each sample by msp2 size polymorphism analysis as previously described [36], and the estimates thus obtained are considered to represent the minimal number of variants present in each sample. Multiplicity of infection was highest in Dielmo, with 64% of isolate samples harbouring a minimum of two different msp2 alleles, followed by Ndiop (40%) Cambodia (16.6%) and Sri Lanka (0%), and correlated with the relative transmission intensity described for each location [34,37]. Polymerase chain reaction (PCR) products of the msp4 and msp5 genes were sequenced directly with a minimum 2.5× coverage. The sequence data was compiled and analysed using ABI® SeqScape® software, which is capable of reading multiple sequences over polymorphic regions and assigning an identity to only the strongest signal identified in overlapping input sequences, with a high degree of

111

confidence. Only the dominant variant present in each sample was used for these analyses and all singletons were confirmed by repeat PCR amplification and sequencing. In total, msp4 gene polymorphism data in 59 isolates was obtained here, and the sequence variation is documented in Fig. 1A. Of the previously reported 15 amino acid substitutions [24–26], 9 were detected in our samples. Two recently described insertion/deletion polymorphisms were confirmed, one new permutation of the deletion near the EGF-like domain was identified and a further nine unreported amino acid polymorphisms were detected. In total, 33 protein variants were identified, with 1 type arising in Sri Lanka, 9 types in Cambodia and 24 types in Senegal (15 in Ndiop and 11 in Dielmo). No geographical clustering of types or allelic grouping within types was detected in our analysis. In contrast to msp4, msp5 gene polymorphism was found to be negligible (Fig. 1B). Of the 23 polymorphic nucleotide sites identified within MSP4, only 5 (21%) were synonymous single nucleotide polymorphisms (sSNPs) and 11 of the 18 nsSNPs were located within 108 nucleotides at the extreme 5 end of the first exon, as found previously in analysis of several laboratory parasite lines [24] and in isolates from Vietnam [25] and Papua New Guinea (PNG) [26]. This rate of ns SNP over sSNP is reflected in values obtained for dS (sSNP per synonymous site) and dN (nsSNP per non-synonymous site) using the method of Nei and Gojobori [38] with Jukes and Cantor correction, as implemented by the package MEGA v2, [39] (n = 57, total gene dS = 5.495 × 10−6 , dN = 8.702 × 10−6 ; exon 1 dS = 9.102 × 10−6 , dN = 17.228 × 10−6 ; exon 2 dS = 14.317 × 10−6 and dN = 15.757 × 10−6 ) [39]. A slight trend of dN > dS is seen, in particular within the first exon, as is common in P. falciparum genes due to codon bias, and cannot be used alone to infer positive selection.

Fig. 1. Sequence diversity. (A) MSP4 sequence variation of 57 isolates and 3 other samples (lab strain 89F5, and p2 and p3, taken from two patients presenting with malaria at a hospital in Paris) sequenced in this study. All residue numbering, shown along the top is to the reference strain NF54 (Accession No. AF295318). Nucleotide and amino acid sequence identity is shown with a dot, variant nucleotides and resultant amino acid substitutions are shown using single letter code with novel changes highlighted in bold, deleted sequence is shown by a dash and synonymous nucleotide polymorphisms are shown in grey. The ID number of each sample is noted in the first panel with C denoting isolates from Cambodia, D, isolates from Dielmo, Senegal, N, Ndiop, Senegal and S for Sri Lanka. Isolates containing a multiple infection are highlighted in bold. The sights most likely to be under balancing selection are marked with an asterisk (B) MSP5 sequence variation for the 38 samples successfully analysed (isolate number in grey mark samples where the sequence of the central 50 residues of the intron, containing a micro-satalite was not attainable). All residue numbering, shown along the top, is to the reference strain 3D7 (Accession No. AF106476). Specific PCR products for msp4 and msp5 were generated separately by nested PCR using up to 5 ␮L of genomic DNA, 2 units of high fidelity VentR ® DNA polymerase (NEB) in a volume of 50 ␮L in the presents of 200 ␮M of equimolar mixed deoxynucleotide triphosphates, 0.5 ␮M of each primer, 5 ␮L of 10× product buffer and 2 mM additional MgSO4 . First round msp4 amplifications were performed using primers MSP4-BF (5 -CAT GCT TTT TCA ACA CAT TTT A-3 ) and MSP4-BR (5 -TTA TAT TAA AAA AAA AAA ATT ATA C-3 ), second round reactions were performed using the previously published primers MSP4-F and MSP4-R [25]. First round msp5 amplifications were performed using the previously published primers MSP5-F and MSP5-R [25], second round amplifications were performed using primers MSP5-HF (5 -GAA AAA AAA GTT TTG ACT TAT GAA TGG-3 ) and MSP5-HR (5 -TTA TCC GAG GGA TAA TAA GTG TGC-3 ). msp4 and msp5 first round amplifications used the following parameters, an initial denaturation step at 94 ◦ C for 2 min followed by 25 cycles of 94 ◦ C for 30 s, 50 ◦ C for 1 min and 62 ◦ C for 3 min with a final elongation step at 72 ◦ C for 10 min. Each second round amplification used the same initial and terminal steps as above with different cyclic parameters; MSP4 required 25 cycles of 94 ◦ C for 30 s, 48 ◦ C for 1 min and 68 ◦ C for 2 min and MSP5 required 30 cycles of 94 ◦ C for 30 s, 50 ◦ C for 30 s and 68 ◦ C for 2 min. Before direct sequencing, each PCR product was gel extracted using the QiaQuick gel extraction kit (Qiagen). Direct sequencing was performed using BigDyeTM v1.1 (Applied Biosciences). Each product was sequenced to a minimum 2.5× coverage using the nested PCR primers (described above) and two further forward and reverse primers. MSP4-for (5 -GTG GAT AGT TAA ATT TTT AAT AGT AG-3 ) and MSP4-Fint (5 AAA GGT AGT TCA ACC AAG TTC ATC-3 ), MSP4-rev (5 -CAA TAT AAC AAC AAA TAT TGT TAT AAA ATG-3 ) and MSP4-Rint (5 -TAC GAT GGG GTA TGC AAT AGG-3 ). MSP5-for (5 -GTA TTC TAT CAT ATA TTT ATT TTT TCG-3 ) MSP5-rev (5 -CAA AAA CAA TGT AAT ATA AAT AAG AAT AG-3 ) MSP5-Fint (5 -TTA GAC AAT GTA GAT GAT GAG GTG CC-3 ) MSP5-Rint (5 -TTG TTT TAT TTT TTT CGT GTG TGT AC-3 ). Singleton sites where confirmed by duplicate PCR. All sequences were assembled using ABI Prism® SeqScape® software v2.0 set to detect secondary peaks at 80% intensity. Only the dominant sequence from each multiple genome was considered.

112

H.E.J. Polson et al. / Molecular & Biochemical Parasitology 142 (2005) 110–115

Values of nucleotide diversity (π, average number of differences between any two sequences) for the coding regions of the msp4 and msp5 genes were calculated using DNAsp v4 software analysis package [40] (Table 1). The values obtained for each location show a striking difference between the two genes, with values for MSP4 diversity generally 10fold higher than those obtained for MSP5. MSP4 diversity in Senegal (3.28 × 10−3 ) is only moderately higher than values reported in PNG (3.1 × 10−3 ) [26] or calculated here for Cambodia (3.08 × 10−3 ). Due to the nature of statistical tests,

deletions within sequences are treated as “gaps in sequence data” and are not analysed by most population genetics software packages, thus automatically assuming that these changes are neither advantageous nor disadvantageous to the organism. Therefore, the two regions subject to deletion within the MSP4 ORF, involving a total of 11 amino acid positions, were not taken into consideration in this analysis, thus excluding 1 non-synonymous site (with a frequency of four) from the calculation. When each deletion was manually substituted by a string of non-synonymous codons, as described

H.E.J. Polson et al. / Molecular & Biochemical Parasitology 142 (2005) 110–115

113

Table 1 Statistical analysis of msp4 and msp5 data sets Country Location msp4 Senegal, Ndiop Senegal, Dielmo Senegal, Total DC Cambodia Sri Lanka Totala msp5 Senegal, Ndiop Senegal, Dielmo Senegal, Total Cambodia Totala

h

S

Ss

π (×10−3 )

Tajima’s D (P-value)

Fu and Li’s D* (P-value)

Fu and Li’s F* (P-value)

27 14 41 41

15 11 24 25

15 13 16 29

4 8 2 7

3.48 2.97 3.28 5.53

−0.594 (>0.1) −1.274 (>0.1) −0.614 (>0.1) −0.738 (>0.1)

0.015 (>0.1) −1.208 (>0.1) 0.758 (>0.1) −0.013 (>0.1)

−0.202 (>0.1) −1.404 (>0.1) 0.358 (>0.1) −0.310 (>0.1)

12 4

9 1

9 0

3 0

3.08 0

−0.035 (>0.1) na

0.108 (>0.1) na

0.081 (>0.1) na

143

49

23

7

3.33

−0.795 (>0.1)

na

na

19 10 29

1 2 2

0 1 1

0 0 0

0 0.54 0.23

0 0.819 (>0.1) −0.387 (>0.1)

0 0.804 (>0.1) 0.598 (>0.1)

0 0.897 (>0.1) 0.376 (>0.1)

8

3

2

2

0.53

−1.310 (>0.1)

−1.409 (>0.1)

−1.513 (>0.1)

71

7

5

2

0.36

−1.602 (>0.05)

na

na

n

All msp4 sequence diversity data is shown in the upper half of the table and msp5 data is shown below, location by location, n: number of samples, h: haplotypes, S: segregating sites, Ss: singleton sites, π: average nucleotide diversity. Taijma’s and Fu and Li’s neutrality tests were implemented by DNAsp. V.4 and validated using the Fisher exact test as implemented by the software. The msp4 deletion corrected (DC) data has been modified as described by Benet et al. (2004) [26]. a Data includes all sequences released in GenBank at the time of analysis and sequences generated in this study for laboratory strain 89F5 (msp4 only), and two patients, p2 (msp4 and msp5) and p3 (msp4 only) presenting with malaria at a hospital in Paris.

by Benet et al. [26] and shown in Table 1 (DC, deletion corrected), these regions could be incorporated into the analysis, greatly increasing the values of diversity obtained (SenegalDC π = 5.53 × 10−3 ). This value is still 10–20-fold smaller than values of π reported for other leading vaccine candidates [5] PfAMA1 (16.35 × 10−3 ), MSP1 (87.92 × 10−3 ), MSP2 (44.09 × 10−3 ) and MSP3 (96.94 × 10−3 ), indicating that MSP4 is a relatively well-conserved surface antigen. To detect any departure from neutrality (the presence of selection) within the data sets generated here, Tajima’s D and Fu and Li’s D* and F* were calculated, (Table 1). Tajima’s D measures the difference between π, the observed average pair-wise nucleotide diversity, and θ, the expected nucleotide diversity under neutrality derived from the number of segregating sites (S). Positive values reflect the presence of high numbers of medium frequency polymorphisms (suggesting balancing selection) and negative values signify a high number of low-frequency polymorphisms (which can be indicative of a growth in population size or a recent selective sweep). Fu and Li’s D* and F* values reflect the same trends by comparing values of θ based on S or the number of singleton nucleotide alleles (D*) or values of θ based on S or the value of π (F*). Despite the greater level of average diversity found within the samples analysed here to those from PNG, our findings are in agreement with Benet et al. [26], and while values tended towards the negative, no significant shift from neutral evolution is seen using any of these tests (Table 1). The negative trend seen here will have been influenced by the high number of singletons present in the data sets and may reflect that malaria parasite populations in Africa have undergone long-term expansion in population size [41,42]. To perform the McDonald–Kreitman test (MK), which is less sensitive to non-equilibrium conditions in data sets,

both the MSP4 and MSP5 P. reichenowi orthologous gene sequences were compiled from shotgun read data available from the Sanger Centre (http://www.sanger.ac.uk/cgibin/blast/submitblast/p reichenowi). Blast searches were preformed using the B8 gene sequences (Accession No. AF033037). Both PrMSP4 and PrMSP5 gene sequences were compiled and found to be 91.5 and 96% identical to their P. falciparum orthologues at the nucleotide sequence level and 86.4 and 94.1% identical at the protein sequence level, respectively. The MK test compares the ratio of synonymous (S) to non-synonymous (N) differences within a group of sequences from one species (S/N = Rwithin ) to the ratio of differences of a closely related species (Rbetween /Rwithin ), generating a value defined as the neutrality index (NI). For the MSP5 data sets generated from Senegal (NI = 0, P = 0.21) and sets using all sequences published to date (NI = 0.09, P = 0.05), the NI value approached 0 due the near absence of non-synonymous polymorphisms found in this gene, although these values do not reach statistical significance. The MSP4 data (Senegal NI = 6.57, P = 0.082; all sequences published to date NI = 0.897, P = 1) shows a trend towards an excess of non-synonymous polymorphisms in P. falciparum, which is particularly evident in the Senegal data set. However, once again these values do not reach statistical significance. Previous analysis of MSP4 diversity in PNG using sliding window analysis of Tajami’s and Fu and Li’s neutrality tests, revealed potential negative selection occurring between nucleotides 106 and 150 of the MSP4 ORF, and non-significant positive values within regions 172–221 and 715–784 [26] (implicating the polymorphic amino acid residues 58, 74, 190 and 213 in possible balancing selection). To investigate the possibility that only certain sites within the MSP4 sequence are under selection, which is frequently not revealed by whole

114

H.E.J. Polson et al. / Molecular & Biochemical Parasitology 142 (2005) 110–115

sequence analysis where values for different regions average out, site-by-site Fst analysis was performed for polymorphic sites with a common allele frequency of <0.9 in populations from PNG and Senegal. It was considered that Fst values not significantly different from zero might reveal sites most likely to be under balancing selection. All of the four positions mentioned above (within the windows with positive Tajima’s values) were present within each population at high enough frequencies to be incorporated into the analysis, and of these, the amino acid positions 74 and 190 showed very low Fst values (−0.024, P = 0.99 and 0.003, P = 0.43, respectively). Interestingly, site 52 (rather than site 58) was also found to have a very low Fst value (−0.025, P = 0.99). The different analyses described here may shed light on the key features of the MSP4 protein sequence. First, the Nterminal hyper-variable region (residues 45–81) includes two sites (D52N and D74G), which are potentially under balancing selection as indicated by a high Tajima’s D index and low Fst indices. This is weak, but suggestive, evidence of a selective signature that could indicate this part of MSP4 is the target of an effective immune response. Second, with regard to the recent identification of two MSP4 derived peptides capable of specifically binding hepatocytes [33], the first (residues 76–92) harbours one semi-conservative polymorphism (at position 81), and the second (residues 113–135) contains one deletion (positions 115–119) or one semi-conservative polymorphism (at position 119). In the existing MSP4 data sets, the alleles at these positions are present at relatively low frequencies. If these latter sites are functionally important due to a role in hepatocyte binding (implying a role for MSP4 in sporozoite invasion) there could be negative selection acting against functionally “less-fit” alleles that would keep them at low frequencies within the population. Clearly, these allelespecific hypotheses, relating to particular sites of MSP4, remain to be tested by immunological and cell binding assays and at present are purely speculative. In contrast, we have confirmed that there is virtually no polymorphism in the msp5 gene, and believe that the crude difference between these gene paralogues reflects a difference in function and/or cellular location. These antigens clearly deserve further investigation regarding the association of specific immune responses with protection from disease in endemic regions, and their suitability as components of a multi-valent malaria vaccine. Acknowledgements This work was funded by the European Malaria Vaccine Development Consortium (QLK2-CT-2002-01197) and all work was carried out at the Institut Pasteur, Paris, with the technical assistance of Sandrine Rosario. We would like to extend our gratitude to Dr. Christophe Rogier and Dr. Shiroma Handunnetti for providing us with isolate samples from Senegal and Sri Lanka, respectively, and give a special note of gratitude to all the inhabitants of the villages Ndiop and Dielmo, for their invaluable participation in site studies.

References [1] Cortes A, Mellombo M, Benet A, Lorry K, Rare L, Reeder JC. Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from the Wosera region of Papua New Guinea. Exp Parasitol 2004;106(1–2):22–9. [2] Genton B, Al-Yaman F, Betuela I, et al. Safety and immunogenicity of a three-component blood-stage malaria vaccine (MSP1, MSP2 and RESA) against Plasmodium falciparum in Papua New Guinean children. Vaccine 2003;22(1):30–41. [3] Stowers AW, Kennedy MC, Keegan BP, Saul A, Long CA, Miller LH. Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against bloodstage malaria. Infect Immun 2002;70(12):6961–7. [4] Hughes MK, Hughes AL. Natural selection on Plasmodium surface proteins. Mol Biochem Parasitol 1995;71(1):99–113. [5] Escalante AA, Lal AA, Ayala FJ. Genetic polymorphism and natural selection in the malaria parasite Plasmodium falciparum. Genetics 1998;149(1):189–202. [6] Volkman SK, Hartl DL, Wirth DF, et al. Excess polymorphisms in genes for membrane proteins in Plasmodium falciparum. Science 2002;298(5591):216–8. [7] Rayner JC, Corredor V, Feldman D, et al. Extensive polymorphism in the Plasmodium vivax merozoite surface coat protein MSP-3alpha is limited to specific domains. Parasitology 2002;125(Pt 5):393–405. [8] 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(11):4382–8. [9] Crewther PE, Matthew ML, Flegg RH, Anders RF. Protective immune responses to apical membrane antigen 1 of Plasmodium chabaudi involve recognition of strain-specific epitopes. Infect Immun 1996;64(8):3310–7. [10] Healer J, Murphy V, Hodder AN, et al. Allelic polymorphisms in apical membrane antigen-1 are responsible for evasion of antibodymediated inhibition in Plasmodium falciparum. Mol Microbiol 2004;52(1):159–68. [11] Marshall VM, Silva A, Foley M, et al. A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain. Infect Immun 1997;65(11):4460–7. [12] Wu T, Black CG, Wang L, Hibbs AR, Coppel RL. Lack of sequence diversity in the gene encoding merozoite surface protein 5 of Plasmodium falciparum. Mol Biochem Parasitol 1999;103(2):243–50. [13] Wang L, Black CG, Marshall VM, Coppel RL. Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum. Infect Immun 1999;67(5):2193–200. [14] Wang L, Richie TL, Stowers A, Nhan DH, Coppel RL. Naturally acquired antibody responses to Plasmodium falciparum merozoite surface protein 4 in a population living in an area of endemicity in Vietnam. Infect Immun 2001;69(7):4390–7. [15] Black CG, Wang L, Hibbs AR, Werner E, Coppel RL. Identification of the Plasmodium chabaudi homologue of merozoite surface proteins 4 and 5 of Plasmodium falciparum. Infect Immun 1999;67(5):2075–81. [16] Black CG, Coppel RL. Synonymous and non-synonymous mutations in a region of the Plasmodium chabaudi genome and evidence for selection acting on a malaria vaccine candidate. Mol Biochem Parasitol 2000;111(2):447–51. [17] Kedzierski L, Black CG, Coppel RL. Characterization of the merozoite surface protein 4/5 gene of Plasmodium berghei and Plasmodium yoelii. Mol Biochem Parasitol 2000;105(1):137–47. [18] Kedzierski L, Black CG, Goschnick MW, Stowers AW, Coppel RL. Immunization with a combination of merozoite surface proteins 4/5 and 1 enhances protection against lethal challenge with Plasmodium yoelii. Infect Immun 2002;70(12):6606–13. [19] Kedzierski L, Black CG, Coppel RL. Immunization with recombinant Plasmodium yoelii merozoite surface protein 4/5

H.E.J. Polson et al. / Molecular & Biochemical Parasitology 142 (2005) 110–115

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

protects mice against lethal challenge. Infect Immun 2000;68(10): 6034–7. Kedzierski L, Black CG, Stowers AW, Goschnick MW, Kaslow DC, Coppel RL. Comparison of the protective efficacy of yeastderived and Escherichia coli-derived recombinant merozoite surface protein 4/5 against lethal challenge by Plasmodium yoelii. Vaccine 2001;19(32):4661–8. Wang L, Goschnick MW, Coppel RL. Oral immunization with a combination of Plasmodium yoelii merozoite surface proteins 1 and 4/5 enhances protection against lethal malaria challenge. Infect Immun 2004;72(10):6172–5. Goschnick MW, Black CG, Kedzierski L, Holder AA, Coppel RL. Merozoite surface protein 4/5 provides protection against lethal challenge with a heterologous malaria parasite strain. Infect Immun 2004;72(10):5840–9. Marshall VM, Tieqiao W, Coppel RL. Close linkage of three merozoite surface protein genes on chromosome 2 of Plasmodium falciparum. Mol Biochem Parasitol 1998;94(1):13–25. Wang L, Marshall VM, Coppel RL. Limited polymorphism of the vaccine candidate merozoite surface protein 4 of Plasmodium falciparum. Mol Biochem Parasitol 2002;120(2):301–3. Jongwutiwes S, Putaporntip C, Friedman R, Hughes AL. The extent of nucleotide polymorphism is highly variable across a 3-kb region on Plasmodium falciparum chromosome 2. Mol Biol Evol 2002;19(9):1585–90. Benet A, Tavul L, Reeder JC, Cortes A. Diversity of Plasmodium falciparum vaccine candidate merozoite surface protein 4 (MSP4) in a natural population. Mol Biochem Parasitol 2004;134(2):275–80. Le Roch KG, Zhou Y, Batalov S, Winzeler EA. Monitoring the chromosome 2 intraerythrocytic transcriptome of Plasmodium falciparum using oligonucleotide arrays. Am J Trop Med Hyg 2002;67(3):233–43. Bodescot M, Silvie O, Siau A, et al. Transcription status of vaccine candidate genes of Plasmodium falciparum during the hepatic phase of its life cycle. Parasitol Res 2004;92(6):449–52. Bottius E, BenMohamed L, Brahimi K, et al. A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes. J Immunol 1996;156(8):2874– 84. Wang L, Menting JG, Stowers A, Charoenvit Y, Sacci Jr JB, Coppel RL. Antigens cross reactive with Plasmodium falciparum merozoite

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41] [42]

115

surface protein 4 are found in pre-erythrocytic and sexual stages. Mol Biochem Parasitol 2000;109(2):189–94. Perlaza BL, Arevalo-Herrera M, Brahimi K, et al. Immunogenicity of four Plasmodium falciparum preerythrocytic antigens in Aotus lemurinus monkeys. Infect Immun 1998;66(7):3423–8. BenMohamed L, Thomas A, Druilhe P. Long-term multiepitopic cytotoxic-T-lymphocyte responses induced in chimpanzees by combinations of Plasmodium falciparum liver-stage peptides and lipopeptides. Infect Immun 2004;72(8):4376–84. Puentes AG, Vera J, Oopez R, et al. Liver stage antigen Plasmodium falciparum peptides bind specifically to human hepatocytes. Vaccine 2004;22:1150–6. Fontenille D, Lochouarn L, Diagne N, et al. High annual and seasonal variations in malaria transmission by anophelines and vector species composition in Dielmo, a holoendemic area in Senegal. Am J Trop Med Hyg 1997;56(3):247–53. Rogier C, Ly AB, Tall A, Cisse B, Trape JF. Plasmodium falciparum clinical malaria in Dielmo, a holoendemic area in Senegal: no influence of acquired immunity on initial symptomatology and severity of malaria attacks. Am J Trop Med Hyg 1999;60(3):410–20. Contamin H, Fandeur T, Bonnefoy S, Skouri F, Ntoumi F, Mercereau-Puijalon O. PCR typing of field isolates of Plasmodium falciparum. J Clin Microbiol 1995;33(4):944–51. Konate L, Zwetyenga J, Rogier C, et al. Variation of Plasmodium falciparum msp1 block 2 and msp2 allele prevalence and of infection complexity in two neighbouring Senegalese villages with different transmission conditions. Trans R Soc Trop Med Hyg 1999;93(Suppl. 1):21–8. Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 1986;3(5):418–26. Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 2001;17(12):1244–5. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 2003;19(18):2496–7. Joy DA, Feng X, Mu J, et al. Early origin and recent expansion of Plasmodium falciparum. Science 2003;300(5617):318–21. Hartl DL. The origin of malaria: mixed messages from genetic diversity. Nat Rev Microbiol 2004;2(1):15–22.