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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Invasion of erythrocytes is a complex, multi-step process and P. falciparum and P. vivax erythrocyte invasion pathways clearly differ in specificity. Plasmodium vivax merozoites invade only reticulocytes,3 whereas P. falciparum merozoites can invade mature erythrocytes as well as reticulocytes.4,5 However, many of the invasion ligands used by the two species are related, supporting the hypothesis that fundamental biologic steps in the invasion process are conserved. The P. vivax Duffy binding protein (PvDBP), which adheres to the Duffy antigen receptor for chemokines on the surface of erythrocytes, is a homolog of erythrocyte binding antigen-175 (PfEBA-175), the P. falciparum ligand that binds to glycophorin A.6–9 Similarly, orthologs of apical membrane antigen 1 (AMA1), a vaccine candidate in both Plasmodium species, appear to be involved in re-orienting the parasite during invasion.10–12 Geographic diversity in these vaccine candidates has been studied extensively, with specific emphasis on functionally important sub-domains.13–15
Members of the reticulocyte binding-like (RBL) superfamily of invasion proteins have been identified in rodent, simian, and human Plasmodium parasites.16–19 The gene family is named after the P. vivax reticulocyte binding proteins 1 and 2 (PvRBP1 and PvRBP2), which bind specifically to reticulocytes and have been proposed to select them for invasion.16,20,21 Homologs have been characterized in P. falciparum,18,22,23 and are referred to as both normocyte binding proteins (PfNBPs) as originally proposed20 and RBP homologs (PfRHs).22,24 The former nomenclature is used in the remainder of this paper. PfNBP1, PfNBP2a, and PfNBP2b have been shown to be involved in invasion, with PfNBP1 binding directly to an unidentified trypsin-resistant receptor on the erythrocyte surface and PfNBP2a and PFNBP2b playing non-overlapping roles during invasion.23,24
This paper presents the first assessment of DNA sequence diversity in P. falciparum and P. vivax RBL genes from Plasmodium isolates originating from geographically distinct locations. Given the potential for important structural and functional domains throughout these proteins, the RBL genes, which range in size from 7.7 to 10 kb, were sequenced and analyzed in their entirety. The presence of related RBL genes in P. falciparum and P. vivax allowed us to compare diversity and selection pressures both within and between Plasmodium species, with surprising results. Diversity levels were generally comparable to other genes involved in erythrocyte invasion, but polymorphism in PvRBP2 was exceptionally high and PvRBP2 sequences could be assigned into two dimorphic classes of alleles. Clustering of polymorphisms was observed in several genes, suggesting that different selection pressures may be acting on different domains. The implications for RBL function, evolution, and consideration as vaccine candidates are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmodium vivax strains and extraction of DNA. The Belem strain was isolated from a patient in Belem, Brazil in 1982 by direct inoculation of infected blood into a splenectomized Saimiri boliviensis monkey (Arruda M, Gysin J, unpublished data). The Thai-NYU strain was established in 1982 in S. boliviensis monkeys by the direct inoculation of sporozoites from infected mosquitoes fed on patients in Thailand. The Thai III strain of P. vivax was established in chimpanzees and New World monkeys from infected mosquitoes fed on a P. vivax-infected individual.26 The Salvador I (Sal I) strain was isolated in La Paz, El Salvador27 and has been passaged in Aotus and Saimiri monkeys and chimpanzees. DNA was prepared using a QIAamp DNA blood mini kit (Qiagen).
Plasmodium falciparum NBP amplification and cloning. PfNBP1. The polymerase chain reaction (PCR) products were amplified using either the Expand High Fidelity PCR System (Roche, Indianapolis, IN) or Pfx DNA Polymerase (Invitrogen, Carlsbad, CA). The complete gene was cloned in five or six overlapping fragments using either a TA cloning kit (Invitrogen) or PCR-Script AMP cloning kit (Stratagene, La Jolla, CA). The primers used in amplifications are as follows: 1F (5'-GATGTATATTTTGCTTATTCTTATTTG-3') and 1R (5'-ACTGCTCGAGTTGTTGAATATAGGTTTG-3'); 2F (5'-GCTAAAGCTTTGAAGAGCACATCAAAC-3') and 2R (5'-GTTCCGTATATATACTAACATG-3'); 3F (5'-CCAAAATGGATGTCGTC-3') and 3R (5'-CTTTTGAAAGAGCGCTTCTTCG-3'); and 4F (5'-CGATAAAACATTTGAAGAACA-3') and 4R (5'-GTCTACTAAAAGGATTGTAC-3'). For some samples, 3F was paired with 4R; 5F (5'-GAGGAC-TATAACTTGTTAC-3') and 5R (5'-CATATCATGT-TCATTCAATG-3'); and 6F (5'-CAAAGTATAGAACAA-CATG-3') and 6R (5'-CTTCGTCAACAGCTTCTA-3').
PfNBP2a and PfNBP2b. Given the extensive region of sequence identity between these two genes,18,22 PfNBP2a and 2b could not be amplified in small fragments because such fragments could not be assigned unequivocally to one gene or the other. Instead complete PfNBP2a and PfNBP2b genes were amplified with a combination of shared and gene-specific primer pairs using the Expand Long Template PCR system (Roche). The primers used were SF9BM (5'-TTCAGGATCCGAGAGCTCTATATGAGTATG-3') and N2ARBM (5'-TTCAGGATCCGATCCACATACCT-GATTG-3') for PfNBP2a and SF8BM (5'-TTCAGGATC-CGCATGAGATTATAGTATAGC-3') and N2BRBM (5'-TCCAGGATCCCATTTATATTGTGTGTATTCC-3') for PfNBP2b. Complete PfNBP2a and PfNBP2b genes were gel purified and digested with Eco RI (there are two internal Eco RI sites in the region shared by both PfNBP2a and PfNBP2b) and Bam HI (Bam HI sites were included in all primers). This digest split each gene into three fragments, which were cloned into Eco RI- (for the internal fragment) or Eco RI/Bam HI (for the two end fragments)-digested pBluescript (Strata-gene) using T4 DNA Ligase (New England Biolabs, Beverly, MA).
Plasmodium vivax RBP amplification and cloning. Complete PvRBP1 and PvRBP2 genes were amplifed as eight or five overlapping fragments, respectively, using the Expand High Fidelity PCR system (Roche). Fragments were cloned using the TA cloning system (Invitrogen).
PvRBP1. Primers used in PvRBP1 amplification were as follows: 1.0FP (5'-CTTACATCTCAGGAAGGCAGAT-3') and 1.0RP (5'-TTCCTTATCACCCAACGTCTCTG-3'); 1.1FP (5'-TCTATATCTTCCCACTTGGGGGC-3') and 1.1RP (5'-TTGCTCTTCTGTTTCTTCCCTGG-3'); 1.2FP (5'-TTAGCGATGACAAGCTGACAGATG-3') and 1.2RP (5'-GCTGACTAAAACGGACAGACTCGC-3'); 1.3XFP(5'-TCAGCAGGTTAACATGAATTTGC-3') and 1.3XRP(5'-GCACGCTCGAACATTCGCTT-3'); 1.4FP (5'-AGGAAGCGAAATAAACGCCTTG-3') and 1.4RP (5'-TCTCCCCTCTTCTGAATGGCAG-3'); 1.5FP (5'-AAACGCTGGAAGAAATTGACCG-3') and 1.5RP (5'-CGCCATTATTAAGCTCCTCTTGGTC-3'); 1.6FP (5'-GAGTTGGAGAGAGAAGCGAACG-3') and 1.6RP (5'-CTCAATATCAACAAAAGAATCATC-3'); and 1.7FP (5'-GAGCATGAATAATGATCCCACGC-3') and 1.7RP (5'-GGAAGTAATGTCTACATGCATGTG-3').
PvRBP2. Primers used in PvRBP2 amplification were as follows: 2.1FP (5'-GATGATCAATTTTTATGCCTGAC-3') and 2.1RP (5'-CAGAATCCGCAATAATAGAG-3'); 2.2FP (5'-CACCCTTATGGGTTCTGAACA-3') and 2.2RP (5'-ACTTCATCATTCATCAAATTA-3'); 2.3FP (5'-AAAAGAAAAGCATAGAAAAAG-3') and 2.3RP (5'-CCTATTTCTTCTAACTTTTCC-3'); 2.4NFP (5'-GGTGAAAGACGACCAATCTAAT-3') and 2.4NRP (5'-AATGTCCGAAATGAAGTTCTG-3'); and 2.5FP (5'-GTGAAAAATCATGGTGATGACC-3') and 2.5RP (5'-CGATATGGGTCTATGGGAAG-3').
Sequencing of DNA. Sequencing was performed using a BigDye Terminator Cycle Sequencing v2.0 Ready Reaction kit (ABI Prism; Applied Biosystems, Foster City, CA) on a 3100 Genetic Analyzer (ABI Prism). Sequences were assembled using Assembly LIGN (Accelrys, San Diego, CA) and all polymorphisms were checked in two or more independent clones for verification. Primer sequences were omitted from the final alignments. Sequence data from this article have been deposited with the European Molecular Biology Laboratory/GenBank Data Libraries under accession numbers as follows: P. falciparum NBP1 sequences, AF411929-33 and AF533700; P. falciparum NBP2a and 2b sequences, AY138496-503; P. vivax RBP1 sequences, AY501884-6; and P. vivax RBP2 sequences, AY501887-9.
Gene analysis.
Sequences were aligned using CLUSTALX 1.8128 and edited in GeneDoc 2.6.002.29 Sequence diversity 
(average pairwise diversity between sequences) and d (diversity corrected for bias in transition-transversion ratios) were calculated for the entire coding sequence of each of the RBL members using DnaSP 3.5330 and MEGA 2.1,31 respectively. Synonymous and non-synonymous substitution rates were calculated using DnaSP 3.53.
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RESULTS |
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). Most of the observed polymorphisms were dimorphic, with one trimorphic site, and polymorphisms tended to be clustered, with the 3' region being the most conserved (Figure 1). Synonymous mutations are relatively rare in P. falciparum genes, a feature that has been used to argue that all extant P. falciparum populations share a recent common ancestor,32 yet 22.5% of the PfNBP1 polymorphisms were synonymous and eight of the nine synonymous mutations were clustered in a region spanning only 1.7 kb (Figure 1).
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The DNA diversity for PfNBP1, estimated using , the average nucleotide diversity between sequences, and d, which corrects for bias in transition-transversion ratios, was 0.00194 and 0.00195, respectively. A pair of PfNBP1 alleles therefore differed on average at only 0.19% of their nucleotide positions. Amino acid repeats that differ in copy number among isolates are common in Plasmodium proteins, and three were noted in PfNBP1 (Figure 1
): an HNx repeat near the C-terminus reported previously23 (x = 4, 6, or 9), and two others, Q(K/T)x (x = 5, 7, 8, or 9), and DIDEINx (x = 3, 4, or 5). A variable stretch of poly-asparagine was also found with five of the strains having 5 Ns at this site, whereas the Brazilian strain 7G8 has 10.
High conservation of PfNBP2aand PfNBP2b.
The PfNBP2a and PfNBP2b genes are related in an unusual manner, with nearly 8 kb of almost identical sequence (referred to as the shared domain), followed by a series of repeats and 3' domains that are unique to each gene.18 To ensure that we were characterizing contiguous PfNBP2a or PfNBP2b genes, the full-length 9.5-10-kb genes were amplified separately by long-range PCR, gel purified, and cloned in fragments (see Materials and Methods). PfNBP2a and PFNBP2b cloned in this manner from four P. falciparum isolates, FVO (Vietnam), 3D7 (Africa), Dd2 (southeast Asia), and 7G8 (Brazil), were sequenced in their entirety and each strain represented a distinct haplotype. Twenty-seven polymorphic nucleotides were identified in PfNBP2a, with six in the series of repeats that marks the boundary between the shared and unique regions, while 23 polymorphisms were identified in PfNBP2b, none of which were in the repeat domain. Diversity in PfNBP2a and PfNBP2b is therefore slightly lower than in the PfNBP1 gene, with values of 0.00159 and 0.00132, respectively (Table 1). In both cases, the polymorphic residues clustered at the 5' end of the gene (Figure 1). All of the PfNBP2b polymorphisms were non-synonymous and only one PfNBP2a polymorphism was synonymous (Table 1). Although the repeats that mark the boundary between the shared and unique regions of these proteins vary in number and repeat pattern between strains, only one gene, PfNBP2a from the Brazilian isolate 7G8, had a repeat sequence that differed from the two versions already described.18 A poly-asparagine tract (7, 8, or 9 Ns) was the only other polymorphic region in the coding sequence.
Twenty-one of the PfNBP2a polymorphisms were in the region that is present in both genes (Figure 1) and 20 of these same polymorphisms were found in the PfNBP2b sequences. Remarkably for 18 of these polymorphic sites, both PfNBP2a and PfNBP2b in a given strain always shared the same nucleotides (Figure 1). For example, at amino acid position 249, both PfNBP2a and PfNBP2b from the Brazilian strain 7G8 encode an asparagine, while both genes from the other three strains all encode a lysine. Similarly, both of the genes in the FVO and 7G8 strains encode a glycine at amino acid position 269, while both genes in the 3D7 and Dd2 strains encode a serine. Of the four polymorphic nucleotides in the shared regions that were not consistent between both genes in a single strain, three are present near the repeated motifs that mark the division between the shared and unique regions, and one is in the short exon 1, which encodes a signal sequence (Figure 1).
Limited polymorphism in PvRBP1 and dimorphism with a high degree of polymorphism in PvRBP2.
The complete PvRBP1 gene was sequenced from four strains (Belem, Thai-NYU, Sal I, and Thai III). Each strain contained a distinct haplotype, with the Belem and Thai-NYU haplotypes being the most similar, differing at only three sites. Within the 8,499-basepair (bp) coding sequence there were 28 polymorphic sites, resulting in diversity estimates comparable to PfNBP1, PfNBP2a, and PfNBP2b, with and d values of 0.00182 and 0.00183, respectively (Table 1). Twenty-five of the 28 polymorphisms were non-synonymous, and 18 of these (67% of the total polymorphisms) cluster within a 1,748-bp region (21% of the coding sequence) near the 5' end of exon 2, similar to the clustering of polymorphism observed in the PfNBP2a and 2b genes (Figure 1).
PvRBP-2 exhibited a significantly greater degree of polymorphism compared with the other RBL genes. Only three haplotypes were identified in four complete PvRBP2 sequences, with the Belem and Thai-NYU isolates containing identical alleles, despite their widely different geographic origins (Brazil and Thailand, respectively). To confirm that the identity between Belem and Thai-NYU PvRBP1 sequences was not the result of sample error, other gene sequences known to differ between the Belem and Thai-NYU strains were amplified from the same sources of genomic DNA and found to differ in sequence as expected. Furthermore, haplotype patterns also suggest that Belem and Thai-NYU isolates are closely related.34 However, the Belem/Thai-NYU PvRBP2 allele differed radically from the PvRBP2 alleles found in the other two isolates, Sal I and Thai-III (derived from El Salvador and Thailand, respectively), which were similar to each other but not identical. At the deduced amino acid level, PvRBP2 sequences are 96.1% identical between Sal I and Thai-III, whereas the Belem/Thai-NYU PvRBP-2 sequence is 83.5% and 84.7% identical to Sal I and Thai-III, respectively. The PvRBP2 sequences could therefore be split into two distinct classes, the Belem/Thai-NYU class and the Sal I/Thai-III class. This apparent dimorphism is shown in Figure 2, both by clustal alignment of the first 800–811 amino acid residues of the encoded PvRBP2 (Figure 2A) and schematically (Figure 2B). There are three regions that are conserved between the two classes, corresponding to nucleotide positions 1294–1574, 7054–7805, and 8367–8604 of the Belem coding sequence (unshaded regions in Figure 2B), but the majority of the encoded protein diverges significantly. Southern blot16 and other unpublished data have shown that the PvRBP2 gene is present as a single copy in the P. vivax genome (Belem strain) and in multiple strains of P. cynomolgi (Okenu DM and others, unpublished data), a simian parasite closely related to P. vivax. These data support the likelihood that the alleles we report here represent a single PvRBP2 gene, rather than sequences from two closely related but divergent PvRBP2 paralogs.
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and d values of 0.04808 and 0.05063, respectively (Table 1). Although the Sal I and Thai-III alleles are much more similar to each other than they are to the Belem/Thai-NYU sequence, there are still 147 nucleotide differences between these two alleles, 107 of which are non-synonymous and 40 are synonymous. The differences between the Sal I and Thai-III PvRBP2 sequences tended to occur in the regions that were otherwise more conserved between all three haplotypes (Figure 2B). Overall, the Sal I and Thai-III PvRBP2 alleles differ at 1.7% of their nucleotide positions ( and d values = 0.01735 [SD = 0.00868] and 0.01759 [SD = 0.00157], respectively). As in PfNBP1, PvRBP2 repeated motifs that varied in number between isolates were also observed. PvRBP2 in the Sal I and Thai-III strains have one or two copies of a PXXQKK motif near the N-terminus, a 24 amino acid deletion near the C-terminus, and a DTHD repeat near the C-terminus, while the Belem and Thai-NYU strains have seven copies of the DTHD repeat.
Selection pressures on RBL genes.
The rates of non-synonymous and synonymous mutations can be used as a measure of whether genes are under selective pressure, with a predominance of non-synonymous mutations implying positive, diversifying selection, whereas a predominance of synonymous mutations would suggest negative, purifying selection. When normalized, the rate of non-synonymous substitutions in PvRBP1, PfNBP2a, and PfNBP2b were all significantly greater than the rate of synonymous substitutions, suggesting diversifying selection (Table 2). In contrast, the rates of synonymous and non-synonymous mutations in PfNBP1 and PvRBP2 were roughly equivalent (Kn/Ks ratios 1; Table 2), suggesting that they are not under significant selective pressure. The synonymous/non-synonymous substitution rates thus differ more significantly between RBL gene family members within each Plasmodium species than they do between the two species, implying that selective pressures are not uniform across RBL genes. However, because the occurrence of synonymous mutations is particularly low in P. falciparum and both non-synonymous and synonymous mutations were found to cluster in different RBL genes, implying that different selective pressures may be acting on different domains, these results should be interpreted cautiously.
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DISCUSSION |
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Polymorphism in merozoite invasion proteins is generally much lower than in merozoite surface proteins (MSPs),36 and is dictated by a balance between the opposite forces of positive selective pressure (such as by the immune system) and purifying functional constraints. Among P. falciparum genes encoding merozoite invasion proteins, PfAMA1 has been the most studied for diversity, with values of 0.0164237 and 0.01402.38 Studies of PfEBA-175 have concentrated on the region encoding the erythrocyte binding domain, Region II, with values for Region II of 0.0036636 and 0.003.14 The homologous region of PfEBA-140/BAEBL, an erythrocyte invasion antigen related to PfEBA-175 that binds to glycophorin C, has a lower value of 0.001.14 The PfNBP genes, with values of 0.00132-0.00194, therefore appear less diverse than either PfAMA1 or the region encoding the PfEBA-175 binding domain, but are similar to the PfEBA-140 Region II.
Fewer analogous studies have been performed using P. vivax genes. A large global study of a 461-bp fragment representing the more variable first half of PvAMA1 produced a value of 0.0174,39 similar to values for PfAMA1. A recent study on the PvDBP gene analyzed diversity in distinct regions and found values ranging from 0.0184 in the erythrocyte binding domain to 0.0086 in Region IV.15 PvRBP1, with a value of 0.00175, is 10-fold less diverse than PvAMA1, and 4-fold less diverse than the most conserved region of PvDBP. In contrast, PvRBP2 has an extremely high value of 0.04808, comparable with the variable block 4 of the P. vivax MSP1 ( value = 0.0451).39 However, of the four PvRBP2 sequences analyzed, two are identical while the other two are much more closely related to each other than either is to the first pair, suggesting that two quite distinct PvRBP2 allelic classes may exist, just as dimorphic alleles exist for both PfMSP1 and PvMSP1.40–42 It is important to note that the two classes of PvRBP2 alleles differ over most of this 9.3-kb gene, whereas in the PvMSP1 or PfMSP1 genes, allelic family differences are restricted to smaller regions. However, the definition of strict classes for PvRBP2 sequences is clearly not completely appropriate. The value between the Thai-NYU and Sal I alleles is still 0.01735, as high as diversity between PvAMA-1 alleles, although it is notably lower than between the Thai-NYU and Thai-III/Belem PvRBP2 alleles (0.06503). In contrast, for PfMSP1 is 0.08792 across 40 sequences of diverse origin, but the value for those sequences decreases to 0.00413 and 0.00100 when comparisons are between members of the same allelic family.36 A more complete geographic analysis, perhaps concentrating on smaller sub-domains of PvRBP2, is necessary to confirm whether there are indeed two or more basic classes of PvRBP2 alleles and whether there is any evidence of recombination between them.
The PfNBP2a and PfNBP2b genes are located on chromosome 13, share more than 8 kb of sequence identity, and are presumed to have arisen as a result of gene duplication.18,22 Our observation that 18 of the 22 polymorphisms in the region shared by both PfNBP2a and PfNBP2b are always the same in both genes in a given strain suggests that recombination and gene conversion are occurring between the two genes. Such intergenic recombination would have the net effect of homogenizing the shared region of the two genes over generations, resulting in parallel evolution and thus explaining the remarkable maintenance of sequence identity. This hypothesis closely mirrors the recent report of a high rate of gene conversion between two closely related falcipain paralogs, falcipain 2A and 2B,43 which lie head-to-tail within 12 kb of each other on chromosome 11, just as the PfNBP2a and PfNBP2b genes lie head-to-tail within 7 kb of each other on chromosome 13.24 The rate of gene conversion in P. falciparum is unknown, but these two reports emphasize that it may be a more powerful force in P. falciparum evolutionary history than has been previously considered. Of the four mutations in the PfNBP2a/2b shared region that are not identical in both genes in a given strain, it is notable that three of them are within 500 basepairs of the repeated sequences that mark the boundary between the shared and unique regions. Crossing over might be less favored at the unique regions because it is more likely to lead to genes with mixed unique domains. The fact that both PfNBP2a and PfNBP2b persist in the P. falciparum genome implies that the two unique domains have distinct functions, so a mixed domain might have a significant functional impact. However, is not yet known whether PfNBP2a and PfNBP2b have distinct or redundant functions, since each can be knocked out independently and at least one strain of P. falciparum lacks one of the two genes, implying that even if they do have distinct functions, they are not both strictly essential.22,24
Sequence analysis can provide information about the nature and force of selective pressures acting on genes, which can in turn provide information about function. Diversifying selection appears to be operating in PfNBP2b, with higher rate of non-synonymous than synonymous substitution and a significant excess of non-synonymous substitutions compared with its ortholog in P. reichenowi. The same trend was seen in PfNBP2a and PvRBP1. Similar selection pressures operating on these genes may reflect their importance in erythrocyte invasion in both parasite species, and may be caused by the human immune response or co-evolution of these ligands with their erythrocyte receptors. Such conclusions could not be drawn significantly for the other RBL genes, given the limited number of sequences generated for each 7.7-10-kb RBL gene in this initial study, which was aimed primarily to assess the general level of diversity over the complete genes. This study provides a clear framework for future investigations involving more isolates, which will be necessary to generate sufficient sample size for detailed population genetic analyses.
The distribution of nucleotide polymorphisms across a gene can also provide clues about function. There is a cluster of eight synonymous mutations in a region of 1.7 kb near the middle of the PfNBP1 gene, for example, which may indicate a domain under purifying selection pressure to minimize diversity. In PfNBP2a, PfNBP2b, and PvRBP1, the majority of the polymorphisms were found within the first 2 kb of exon 2, and most of those substitutions were non-synonymous. This pattern of polymorphism has interesting parallels with PvDBP, where Region II, which contains the erythrocyte-binding domain, is also the most polymorphic region and has the highest ratio of non-synonymous to synonymous substitutions.15 The relatively polymorphic N-terminal regions of PfNBP2a, PfNBP2b, and PvRBP1 are therefore potentially functionally important domains, and initial studies indicate that the N-terminus of PvRBP1 may indeed function as an erythrocyte-binding domain (Rosas-Acosta G and others, unpublished data).
The RBL proteins are candidates for inclusion in erythrocyte invasion-blocking vaccines. In P. vivax, the reticulocyte recognition step predicted to be catalyzed by the PvRBPs is an essential step in the invasion cascade.16,20 Similarly, in P. falciparum, the PfNBPs are predicted to function in the early red blood cell recognition steps.18,20,24 Sequence diversity of potential vaccine constituents is an important concern because polymorphism may limit the ability of a single vaccine to be used globally and also may indicate the likelihood of escape mutants emerging under vaccine-induced immune pressure. This report shows that P. falciparum and P. vivax RBL gene family members are generally highly conserved, with the exception of PvRBP2, in which greater diversity was noted. Ongoing vaccine studies will consider whether the function or immunogenicity of the RBLs is affected by the limited diversity noted here.
Received May 28, 2004. Accepted for publication November 29, 2004.
Financial support: Julian C. Rayner was supported by the Human Frontier Science Program and by the American Society of Microbiology as an American Society of Microbiology/National Center for Infectious Diseases Postdoctoral Research Fellow. This research was supported by grant number R01 AI24710-17 from the National Institutes of Health and by the National Center for Infectious Diseases, Centers for Disease Control and Prevention.
* Both authors contributed equally to this work. 
Authors’ addresses: Julian C. Rayner, Division of Geographic Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35209, Telephone: 205-934-5804, Fax: 205-934-5600, E-mail: jrayner{at}uab.edu. Tuan M. Tran and Mary R. Galinski, Emory Vaccine Center, Yerkes National Primate Research Center and Department of Medicine, Division of Infectious Diseases, Emory University, Atlanta, GA 30329, Telephone: 404-727-7214, Fax: 404-727-8199, E-mails: tmtran{at}emory.edu and galinski{at}rmy.emory.edu. Vladimir Corredor, Departamento de Ciencias Fisiológicas, Facultad de Medicina, Universidad Nacional de Colombia, Bogota, Colombia, E-mail: vcorred{at}rmy.emory.edu. Curtis S. Huber and John W. Barnwell, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, Telephone: 404-488-4528, E-mails: cwh7{at}cdc.gov and wzb3{at}cdc.gov.
Reprint requests: Mary R. Galinski, Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329.
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