DOI: https://doi.org/10.21203/rs.3.rs-1023914/v1
Background
Plasmodium vivax reticulocyte binding protein 2b (PvRBP2b) plays a critical role in parasite invasion of reticulocytes by binding the transferring receptor 1. PvRBP2b is a vaccine candidate since the antibody titers against PvRBP2b recombinant proteins are negatively correlated with the parasitemia and risk of vivax malaria. This study aims to analyze the genetic diversity of the PvRBP2b gene in the global P. vivax populations.
Methods
The near full-length PvRBP2b nucleotide sequences (190-8349 bp) were obtained from 88 P. vivax isolates collected from the China–Myanmar border (n=44) and Thailand (n=44). Additional 224 sequences of PvRBP2b were retrieved from genome sequences from the global parasite populations. The genetic diversity, neutral selections, haplotypes distribution and genetic differentiation of PvRBP2b were examined.
Results
The genetic diversity of PvRBP2b was distributed unevenly with the peak in the reticulocyte binding region in the N-terminus and subjected to the balancing selection. Several amino acid variants were found in all or nearly all endemic fields. However, the critical residues responsible for reticulocyte binding were highly conserved. There was substantial population differentiation according to the geographical separation. The distribution of haplotypes in the reticulocyte binding region varied among regions; even the two major haplotypes Hap_6 and Hap_8 were found in only five populations.
Conclusions
Our data showed considerable genetic variations of PvRBPb in global parasite populations, and the geographic divergence may pose a challenge to PvRBP2b-based vaccine development.
Malaria remains a threat to global health despite intensified control efforts in recent years. As the most widespread Plasmodium species, Plasmodium vivax caused an estimated 7.9 million worldwide in 2018, with 53% of the vivax burden being in the Southeast Asia Region[1]. It is challenging to eliminate P. vivax due to its dormant liver stage, which gives rise to the relapses of malaria [2, 3, 4]. Integrated interventions, including novel tools such as vaccines, are urgently needed for malaria elimination.
Invasion of the red blood cells (RBCs) by the merozoites is an essential step for the asexual erythrocytic cycle of malaria parasites [5, 6]. Since merozoites are exposed to the host immune system, vaccine candidates are usually designed to target the merozoite surface proteins to block erythrocyte invasion [7, 8]. Compared to P. falciparum, much fewer vaccine candidates have been identified for P. vivax, partially owing to the absence of a long-term in vitro culture system for this parasite [9, 10]. P. vivax species requires the Duffy antigen receptor for chemokines (DARC) on the RBC surface for invasion. However, solid evidence of P. vivax infection in the DARC-negative individuals in Africa suggests that P. vivax may have evolved to explore alternative pathways for invasion [11, 12]. P. vivax shows a restricted tropism for reticulocytes with high levels of transferrin receptor 1 (TfR1 or CD71) [13]. Recently, TfR1 has been identified as the reticulocyte-specific receptor for P. vivax reticulocyte-binding protein 2b (PvRBP2b), a ternary complex expressed in the schizont stage [14, 15]. PvRBP2b belongs to the PvRBP family composed of at least 11 members with different binding preferences for normocytes or reticulocytes [5, 16, 17, 18, 19]. The crystal structure of the N-terminal domain of PvRBP2b has revealed a similar structural scaffold to that of P. falciparum reticulocyte-binding protein homolog 5 (PfRh5), a well-characterized vaccine candidate for P. falciparum [15, 20]. Monoclonal antibodies against PvRBP2b or TfR1 mutants that impede the binding of PvRBP2b to the reticulocytes successfully blocked the entry of P. vivax into the reticulocytes, suggesting PvRBP2b as a promising vaccine candidate targeting blood-stage infections [15].
The N-terminal domain of PvRBP2b is responsible for reticulocyte binding [14, 15], whereas the function of the C-terminal domain was not clear. With the recombinant PvRBP2b N-terminal domain, antibodies against PvRBP2b have been detected in P. vivax patient plasma samples, supporting that PvRBP2b contains immune recognition epitopes in the N-terminal domain [15, 18, 21]. Furthermore, the IgG levels against PvRBP2b were negatively correlated with parasitemia and the risk of vivax infections [18, 21, 22]. These studies highlight PvRBP2b as a promising target for P. vivax vaccine development [14, 15].
A major challenge for the efficacy of blood-stage malaria vaccines is the extensive genetic diversity of the target antigens. Therefore, understanding the genetic diversity of vaccine candidates is necessary for designing effective vaccines and predicting vaccine efficacy. In this study, we analyzed the genetic diversity, phylogenetic relationship, and population differentiation of PvRBP2b from 312 global P. vivax isolates, aiming to provide the necessary information for PvRBP2b-based vaccine development.
This study used 88 dried blood spots on filter papers collected from P. vivax patients attending the Laiza and Nabang hospitals in the China–Myanmar border area in 2014 ( n=44) and the Tha Song Yang hospital in western Thailand in 2011-2012 (n=44). Malaria was diagnosed by microscopy, and finger-prick blood samples were collected.
PvRBP2b gene amplification and sequencing
Genomic DNA was extracted from dried blood spots on ଁlter papers using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Of the full-length protein-coding sequence of the PvRBP2b gene (8421bp), an 8160 bp fragment (PvRBP2b190-8349), corresponding to the amino acids 64-2783 of the PvRBP2b protein was amplified from all the samples using KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan). Given the large size of the gene, seven overlapping 1.5 kb fragments were amplified from each sample using seven pairs of primers (Table S1). PCR reactions were performed in 30 µl volume containing 1×KOD-Plus-Neo buffer, 200 µM dNTPs, 1 mM MgSO4, 250 nM primer, 0.4 units KOD Plus polymerase, and 2 µl genomic DNA. The following cycling parameters were used: an initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 15 s, annealing at a determined temperature (Table S1) for 15 s, and extension at 68°C for 90 s, followed by a ଁnal extension at 68°C for 5 min. The PCR products were separated in 1% agarose gels and then subjected to DNA sequencing using the ABI BigDye™ Terminator Reaction Ready kit (Applied Biosystems, CA, USA).
The 88 PvRBP2b sequences were assembled using the DNASTAR program (Lasergene). In addition, 224 PvRBP2b sequences from eleven global locations were obtained from previous whole-genome sequencing projects [23, 24, 25, 26, 27, 28]. Fastq files were downloaded from the Sequence Read Archive (SRA) of the National Center for Biotechnology Information. We used two parameters to exclude low-quality variants: quality ≤40, minor allele frequency less than 0.01. Only SNP variants in single infection were included according to separate criteria. After filtering out unqualified sequences, the global sample set includes Brazil (n=36), Colombia (n=28), Cambodia (n=35), China–Myanmar border (n=19), Ethiopia (n=18), Indonesia (n=3), Laos (n=2), Malaysia (n=4), Papua New Guinea (PNG) (n=20), Thailand (n=48), and Vietnam (n=11). Isolate codes and SRA accession numbers of samples used in this analysis are given in Table S2.
A total of 312 PvRBP2b sequences were aligned with the reference sequence from the Salvador I (Sal I) strain (PVX_094255) using the Clustal W program in the MEGA7 software. For the evaluation of PvRBP2b genetic diversity, the nucleotide diversity (π), the number of haplotypes (H), and haplotype diversity (Hd) were computed using the DnaSP v5.10 software [29]. To test the departure from neutrality, Tajima's D test [30], Fu & Li's F* test [31], and Fu & Li's D* test [31] were computed using DnaSP v5.10 software. The McDonald-Kreitman (MK) test was performed to evaluate the departure from neutrality using the P. cynomolgi sequence (PcyRBP2b; PCYB_081060) as the outgroup [32]. Fisher's exact test was applied to assess statistical significance (p < 0.05).
Natural selection was determined by calculating the ratio of nonsynonymous (dN) to synonymous (dS) substitutions per nucleotide site (dN-dS), using the Nei-Gojobori method [33] with Jukes-Cantor correction for multiple substitutions. Statistical significance of the difference was estimated using the codon-based Z-test of selection implemented in MEGA 7 [33].
Finally, to determine the existence of specific codons targeted by selection in the global population [34], three maximum likelihood codon-based tests in the HyPhy package-based algorithms, SLAC [35], FEL [35], and FUBAR [36] implemented in the Datamonkey webserver (http://www.datamonkey.org) were performed.
To investigate population subdivision, Wright's fixation index (FST) representing inter-population variance in allele frequencies was calculated using DnaSP v5.10 [29, 37, 38]. The genetic structure of all the vivax parasite populations was then elucidated using the STRUCTURE v2.3.2 software based on the Bayesian analysis and admixture model [39, 40]. All samples were run at K = 2–7 (10 iterations each) with a burn-in period of 20000 iterations followed by 1200000 Markov Chain Monte Carlo (MCMC) iterations. Then the optimal number of grouping was determined by ΔK using the STRUCTURE HARVESTER v0.6.94 software [41, 42]. The partition of the clusters was presented using CLUMPP v1.1.2 [43] and the DISTRUCT 1.1 tools [44]. To determine the relationship among the parasites, phylogenetic analyses were performed using the Neighbor-Joining method implemented in MEGA7 [33]. Then the phylogenetic tree was optimized with the online tool ITOL [45]. A haplotype network based on the polymorphic sites in the reticulocyte-binding region of PvRBP2b was constructed using the PHYLOVIZ 2.0 software with the Neighbor-Joining method [46].
The potential linear B-cell epitopes were predicted using the BCPreds prediction tool (http://ailab-projects1.ist.psu.edu:8080/bcpred/predict.html) [47]. Antigenicity was predicted using the VaxiJen v2.0 online tool (http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html) [48]. BCPreds predicts a peptide length of 12 consecutive amino acids with a threshold of 0.8, whereas VaxiJen sets a threshold at 0.5. The overlapped regions of predicted linear B cell epitopes by both methods were selected. The predicted 3D structures of the reticulocyte-binding region of PvRBP2b were constructed with the PHYRE2 algorithm [49] and further visualized and modeled using the molecular modeling tool PyMOL V2.3 [50]. The PvRBP2b amino acid sequence in the reference Sal I strain was used for prediction.
Mutations revealed from global PvRBP2b sequences
Sequencing of the 8160 bp PvRBP2b fragment (190–8349 bp) was successful for the 88 P. vivax field isolates collected from the China–Myanmar and Thailand–Myanmar border areas. To gain a global perspective, we retrieved 224 PvRBP2b sequences from the whole genome sequences of P. vivax isolates collected in multiple P. vivax-endemic areas of the world. Alignment of all 312 PvRBP2b sequences with the Sal I reference identified 116 single nucleotide polymorphisms (SNPs), including 96 nonsynonymous and 20 synonymous mutations. All nonsynonymous mutations with allele frequencies of more than 1% in different areas are shown in Fig. 1 and Table S3. The E136K, N349K, K363E, D366V/H, V395A/T, K412N, Q564R, D917E, N1529K, K1606E, E2265K, and E2746G amino acid mutations were found in all or nearly all endemic sites, reflecting the prevalent PvRBP2b polymorphisms in the world. Among them, D917E approached fixation (95.8%).
The reticulocyte binding region on the N-terminus of PvRBP2b is the critical portion for the receptor binding and RBC invasion. We refer it to the amino acid residues 168-633 since its flanking regions failed to be visualized in the PvRBP2b structure [14]. As shown in Table S3, from a total of 75 nonsynonymous nucleotide substitutions with allele frequencies > 1%, almost 50% (35/75) were accumulated in the reticulocyte binding region, generating 28 nonsynonymous amino acid mutations. Besides the 11 nonsynonymous mutations (R217H, R242T/S, K288P, K309Q, K363E, D366V/H, G382R/E, E497K, D558E, Q564R, and N591K) reported for the reticulocyte binding region [14], 17 additional nonsynonymous mutations (D220Y, T224R/K, S228P, E232K, L293V, N300K/D, D315Y, N349K, V395A/T, K412N, Q413E, K437E, H455Q, K575E, D578H, S586R, and E631K) were reported in this study (Fig. 2a). Among them, K363E and S586R are the residues interacting with the receptor TfR1 [14]. K363E was prevalent in all endemic areas with an allele frequency of 40.4%, whereas S586R was found only in Brazil (22.2%), Ethiopia (11.1%), and Thailand (1.1%) (Table S3).
Genetic diversity of PvRBP2b
We analyzed the population genetic indices to assess the nucleotide diversity of PvRBP2b (Table 1). The overall nucleotide diversity (π) from 312 sequences was 0.00196, with the highest found in the population from Malaysia (0.00203), followed by Thailand (0.00198), and Vietnam (0.00193). The overall haplotype diversity was high (0.997), with the highest found in Malaysia, Vietnam, PNG, Indonesia, and Laos (Table 1). PNG showed much lower nucleotide diversity (0.00114) but high haplotype diversity (1.000) (Table 1). The sliding window plots of nucleotide diversity revealed an uneven distribution with the peaks located at nucleotides 1015–1134 within the reticulocyte binding region (Fig. 3a). Similarly, 46.7% amino acid substitutions were clustered in the reticulocyte binding region (Fig. 3e). This result reflected relatively high polymorphism in the reticulocyte binding region of PvRBP2b in the worldwide P. vivax populations.
| Populations | No. isolates | Polymorphic sites | π ± SD | H | Hd±SD |
|---|---|---|---|---|---|
| Brazil | 36 | 30 | 0.00121±0.00089 | 18 | 0.930±0.023 |
| Colombia | 28 | 29 | 0.00112±0.00091 | 18 | 0.950±0.025 |
| Cambodia | 35 | 55 | 0.00172±0.00167 | 30 | 0.987±0.012 |
| China-Myanmar | 63 | 81 | 0.00178±0.00213 | 45 | 0.973±0.012 |
| Ethiopia | 18 | 32 | 0.00120±0.00114 | 16 | 0.980±0.028 |
| Laos | 2 | 9 | 0.00110±0.00110 | 2 | 1.000±0.500 |
| Malaysia | 4 | 31 | 0.00203±0.00208 | 4 | 1.000±0.177 |
| Papua New Guinea | 20 | 39 | 0.00114±0.00135 | 20 | 1.000±0.016 |
| Thailand | 92 | 97 | 0.00198±0.00244 | 85 | 0.998±0.002 |
| Vietnam | 11 | 43 | 0.00193±0.00184 | 11 | 1.000±0.039 |
| Indonesia | 3 | 16 | 0.00131±0.00131 | 3 | 1.000±0.272 |
| Total | 312 | 147 | 0.00196±0.00299 | 248 | 0.997±0.0008 |
| H: number of haplotypes, Hd: haplotype diversity, SD Standard deviation | |||||
Neutrality tests were conducted to evaluate whether the PvRBP2b gene followed the neutral equilibrium model of molecular evolution. Although the overall PvRBP2b sequence did not significantly deviate from neutrality, a sliding window analysis identified significant positive values for the 1015–1034 bp region by Tajima's D*, and Fu and Li's F* tests, which paralleled the peak π value, reflecting balancing selection within the reticulocyte binding region in the global populations (Fig. 3b-d). In addition, some significant negative values for several C-terminal domains suggested the population expansion or excess of singletons (Fig. 3b-d).
Departure from neutrality was further evaluated using the MK test with the P. cynomolgi RBP2b sequence as the outgroup [32]. The results showed significantly more intraspecific nonsynonymous substitutions over synonymous substitutions than interspecific fixed differences in both the entire sequenced region (NI=1.874, p=0.005753) and reticulocyte-binding region (NI=6.134, p=0.004504) (Table 2). The dN-dS statistic was consistently positive for the reticulocyte-binding region among the global populations (Table 2), suggesting that polymorphisms found for this region of PvRBP2b were maintained by diversifying selection.
| Gene fragment encoding PvRBP2b | N | MK test | Codon-based Z-test | ||
|---|---|---|---|---|---|
| NI | p-value | dN-dS | p-value | ||
| Near full-length | 312 | 1.874 | 0.005753** | 0.995 | 0.322 |
| Reticulocyte binding region | 312 | 6.134 | 0.004504** | 2.801** | 0.006 |
| N: Number of isolates; NI: neutrality index; *, p<0.05 **, p<0.01 | |||||
Three likelihood-based algorithms were used to determine specific codons targeted by selection. Thirty-one positively selected and 12 negatively selected codons were identified by all three algorithms (Table 3). Twelve positively selected mutations are located in the reticulocyte-binding region, presumptively associated with parasite invasion and/or immune recognition (Table 3). K363 and S586 have been shown as the reticulocyte-binding sites [14]. In contrast to the positive selection at K363 confirmed by all three methods, S586 was supposed to be positively selected only by the FUBAR method (Table 3). Meanwhile, sites under purifying selection were scattered along the PvRBP2b (Table 3).
| SLAC | FEL | FUBAR | By all three algorithms | |
|---|---|---|---|---|
| positive/diversifying selection sites | 136, 217, 224, 242, 288, 349, 363, 382, 395, 412, 564, 575, 591, 1181, 1381, 1510, 1529, 1606, 1870, 2073, 2190, 2200, 2221, 2250, 2261, 2265, 2278, 2318, 2741, 2746, 2750 | 136, 217, 220, 224, 242, 288, 293, 315, 349, 363, 382, 395, 412, 413, 558, 564, 575, 591, 917, 1181, 1239, 1381, 1510, 1529, 1577, 1606, 1870, 1984, 2073, 2190, 2200, 2221, 2236, 2250, 2261, 2265, 2278, 2318, 2642, 2741, 2746, 2750 | 136, 217, 220, 224, 228, 232, 242, 288, 293, 300, 309, 315, 349, 363, 382, 395, 412, 413, 437, 455, 497, 558, 564, 575, 578, 586, 591, 631, 666, 917, 1168, 1173, 1181, 1239, 1289, 1381, 1510, 1520, 1529, 1577, 1606, 1870, 1907, 1984, 2073, 2190, 2200, 2201, 2221, 2236, 2250, 2261, 2265, 2272, 2278, 2318, 2382, 2393, 2517, 2605, 2613, 2642, 2738, 2741, 2746, 2747, 2750 | 136, 217, 224, 242, 288, 349, 363, 382, 395, 412, 564, 575, 591, 1181, 1381, 1510, 1529, 1606, 1870, 2073, 2190, 2200, 2221, 2250, 2261, 2265, 2278, 2318, 2741, 2746, 2750 |
| negative/purifying selection sites | 885, 1113, 1644, 1834, 1886, 2149, 2225, 2238, 2247, 2255, 2292, 2329, 2391, 2465 | 243, 670, 885, 1113, 1644, 1834, 1886, 1917, 1987, 2149, 2225, 2238, 2247, 2255, 2292, 2329, 2391, 2465 | 885, 1113, 1644, 1834, 1886, 1987, 2149, 2247, 2255, 2292, 2329, 2391, 2465 | 885, 1113, 1644, 1834, 1886, 2149, 2247, 2255, 2292, 2329, 2391, 2465 |
The 3D model structure with positively selected amino acid mutations mapped in the reticulocyte-binding region of PvRBP2b showed that most mutated residues were on the surface of α helices except V395 hidden inside the protein and K412 located in the flexible loop structure (Fig. 2b). Most mutations were close to the hydrophobic binding region. Three peptides (270–281, 519–530, and 566–577) were predicted to be linear B-epitopes with the prediction scores of 0.839, 0.987, and 0.959, respectively, by the BCPreds and VaxiJen software (Table 4). However, only one polymorphic residue, K575, was presented in the predicted B-epitope of 566–577.
| Start Position | Stop Position | Sequence | Score |
|---|---|---|---|
| 270 | 281 | KLRQYEEKKEAF | 0.839 |
| 519 | 530 | NEFKKDYDNNVE | 0.987 |
| 566 | 577 | NIPANSNAQKKV | 0.959 |
The 17 SNPs with minor allele frequencies of >5% in the PvRBP2b reticulocyte-binding region were chosen for haplotype analysis, which yielded a total of 114 haplotypes amongst the global dataset of 312 sequences (Table S4 and Fig. 4-5). Of the 114 haplotypes, 58 (50.9%) were region-specific, with a frequency between 0.32% and 1.60%. There were many rare haplotypes; 92.1% (105/114) of the haplotypes were shared by no more than five parasite isolates, among which 70/114 were represented by single parasite isolates. Even the two major haplotypes Hap_6 and Hap_8, with a frequency of 14.74% and 12.82%, respectively, were only found in five regional populations. Hap_8, the same as the Sal I haplotype, was most abundant in the China–Myanmar border (39.7%) and Colombia (42.9%). Hap_6, the predominant haplotype identified in Thailand, was also shared among the parasite populations from the China–Myanmar border, Cambodia, Brazil, and Colombia. The haplotypes in Asia were distributed in all global parasite populations, and Thailand harbored the highest haplotype diversity (52/114) (Table S4 and Fig. 4, 5). In contrast, the distribution pattern shown in the haplotype network was different from parasite populations from South America (Brazil and Colombia), Africa (Ethiopia), and Oceania (PNG) (Fig. 5). Sixteen out of 20 parasite isolates in PNG were from a unique haplotype restricted to this specific region (Table S4 and Fig. 4).
Analysis of the PvRBP2b sequences showed that the global parasites were optimally grouped into three clusters (K = 3) (Fig. 6a). These clusters were unevenly distributed among different geographic regions. The parasites from PNG and Indonesia were mainly represented by the purple cluster, whereas the parasites from Ethiopia, Brazil, and Colombia occupied the green cluster. The red cluster represented parasites predominantly from the China–Myanmar border and Thailand. Interestingly, the remaining parasites from the China–Myanmar border and Thailand and parasites from other countries of the Greater Mekong Subregion (GMS) (Cambodia, Laos, and Vietnam) showed genetic mixing of the green and purple clusters. When structure analysis was conducted using the PvRBP2b reticulocyte-binding region, six clusters were identified with overlapping, worldwide distribution (Fig. 6b). It is noteworthy that only the parasites from the PNG and Indonesia formed genetically distinct clusters from other geographic regions.
The genetic differentiation between two parasite populations was also evaluated via Wright's fixation index (FST) using the entire PvRBP2b sequence and reticulocyte-binding region, respectively. The heatmap of the FST values from both analyses revealed population differentiation patterns consistent with the Structure analysis (Fig. 7). Consistent with the principle of isolation by distance, parasite populations were genetically similar within each continent (e.g., Brazil vs. Colombia, PNG vs. Indonesia, countries within the GMS). In contrast, considerable differentiation was detected between populations from different continents. Notably, the PNG and Indonesia P. vivax populations had high levels of genetic differentiation from the rest of the global parasite populations. In comparison, parasite populations from the GMS were moderately differentiated from the South American parasite populations. Interestingly, parasites from Africa (represented by Ethiopia) showed little genetic differentiation from the GMS and South American parasites.
This study evaluated the genetic diversity of PvRBP2b as a P. vivax malaria blood-stage vaccine candidate. The overall nucleotide diversity of PvRBP2b from the global samples was modest (0.00196), much lower than that of the highly polymorphic surface antigens, such as PvAMA1 and PvMSP1 [51, 52]. The genetic polymorphisms of PvRBP2b were geographically heterogeneous, with higher diversity found in the GMS countries than in South America (Brazil and Colombia), Africa (Ethiopia), and Oceania (PNG). Such a genetic diversity distribution pattern was also observed in PvAMA1, and it seemed to correlate with the larger effective population size in Southeast Asia [53]. It is speculated that P. vivax in Southeast Asia was the possible source population [54]. The high transmission intensity and the frequent migrations of infected people among Southeast Asian countries might result in the large effective population size in this continent. Recently, continuous malaria control and elimination strategies had successfully reduced the parasite incidence but seemed to have little short-term effect on the P. vivax population size [55, 56]. The relatively high levels of asymptomatic vivax infections in endemic populations possibly contributed to the maintenance of the effective population size [57, 58].
PvRBP2b-mediated the reticulocyte invasion depends on the PvRBP2b–TfR1–Tf ternary complex [14]. Consistent with the previous reports [15], the reticulocyte binding region was under the balancing selection. It accumulated most of the nonsynonymous amino acid substitutions and had the highest diversity. Interestingly, most of the residues on the reticulocyte binding region were conserved. At least three residues (Y542, K600, and Y604) on PvRBP2b were critical to the reticulocyte binding and complex formation because mutations at these sites reduced binding affinity by around 80% [14]. These amino acids were absolutely conserved among all 312 samples, reflecting their essential function in reticulocyte binding. Most of the identified residues making contact with TfR1/Tf from structure analysis [14] were conserved except S586R and K363E which had low (3.5%) and moderate (40.26%) allele frequencies, respectively. S586R had a limited distribution in Brazil, Ethiopia, and Thailand, whereas K363E occurred in all endemic areas. Additional nonsynonymous SNPs of low-modest frequencies (1.6–51%) were detected surrounding the interaction sites. Several residues within the reticulocyte binding region were under positive selection, raising the possibility that they were the result of the host immune selection [59]. Moreover, most of the positively selected residues are located on the α helices, some being close to the hydrophobic binding region. The K412N mutation had a frequency of 51% and resided in the flexible loop structure. Only one mutation, K575E, was mapped to the predicted linear B-cell epitope. However, it is still unclear whether these specific polymorphic residues would change protein structure by altering protein polarity and hydrophilicity, therefore adapting to the different TfR1 mutants (e.g., L212V, N348A, and S412G) [14, 60, 61]. More functional investigations are required to answer these questions.
One of the obstacles hindering the development of a successful malaria vaccine is the extensive genetic diversity of blood-stage antigens in P. vivax [62]. Naturally acquired antibodies against the reticulocyte binding region of PvRBP2b showed a strong association with the reduced parasitemia [18, 21, 22], highlighting the vaccine potential of PvRBP2b. Although the overall genetic diversity of PvRBP2b was not as high as that of leading blood-stage vaccine candidates, PvAMA1 and PvMSP1, which have advanced to clinical trials [63], the reticulocyte binding region of PvRBP2b deserves further attention. This region carried almost 50% of the mutations in the entire PvRBP2b, and analysis of the global parasite samples identified 114 haplotypes. Furthermore, most haplotypes were region-specific and represented by a single parasite isolate, and very few haplotypes were shared worldwide. Even the predominant haplotypes Hap_6 (14.74%) and Hap_8 (12.82%) were distributed in only five regions. Of note, PNG showed distinct haplotype patterns from other worldwide populations. This enormous haplotype diversity may present a challenge to developing a PvRBP2b-based vaccine. Since an effective vaccine should include most of the common alleles relevant to the induction of immune responses to ensure sufficient coverage of the genetic diversity [64, 65], it is essential to determine whether the major PvRBP2b alleles confer strain-transcending immunity.
This study provides several lines of evidence confirming the geographical separation of global P. vivax populations. Pairwise FST comparison identified the considerably high levels of differentiation between the Oceania (PNG and Indonesia) parasite populations from other endemic regions, whereas parasite populations in the GMS were much less differentiated. Interestingly, populations from South America appeared to be closely related to those from Africa (Ethiopia). Population structure analysis further reinforced such a finding on population relatedness. The population differentiation pattern identified here was in agreement with other population studies using individual genes [51], microsatellites [54, 66], or whole genome sequences [23, 24, 67]. The distinct parasite genetic structure in PNG may reflect the limited gene flow between PNG and the rest of the world, while the unique RBC polymorphisms in the human populations may have also contributed to this difference [53, 66, 68, 69]. In the GMS, however, the high transmission intensity in some border areas and frequent host migrations among the different countries are likely responsible for the panmixia of parasite populations. Population genetics can help assess the effects of malaria control strategies, track the source of imported infections, and inform the vaccine design. Therefore, continuous monitoring of the population genetic structures will allow longitudinal evaluation of the efficacy of the malaria control strategy both locally and globally.
In conclusion, this study revealed a remarkably high level of genetic polymorphisms in the PvRBP2b among the global P. vivax populations, with mutations clustered in the reticulocyte binding region. The genetic differentiation of parasite populations among different continents were remarkable, suggesting a potential need to cover major protein variants in case of strain-transcending immunity. Future studies addressing the functions of antibodies against different PvRBP2b variants are warranted.
RBCs: Red blood cells; DARC: Duffy antigen receptor for chemokines; TfR1: Transferrin receptor 1; PfRh5: P. falciparum reticulocyte-binding protein homolog 5; PNG: Papua New Guinea; PCR: Polymerase chain reaction; SNPs: Single nucleotide polymorphisms; SRA: Sequence Read Archive; π: The nucleotide diversity; H: the number of haplotypes; Hd: haplotype diversity; MK: McDonald-Kreitman; FST: Wright’s fixation index; PvAMA1: Plasmodium vivax apical membrane antigen; PvMSP: Plasmodium vivax Merozoite surface protein 1; GMS: Greater Mekong Subregion.
Acknowledgments
We would like to thank the patients participating in this study and providing the blood samples.
Funding
This work was supported by grants (U19AI089672 and U01AI155361) from the National Institute of Allergy and Infectious Diseases, NIH, USA.
Availability of data and materials
The data supporting the conclusion of this article are included within the article.
Authors’ contributions
XZ performed the experiment study and drafted the manuscript. HW and YMZ, participated in data analisis.YZ, LW, YH and JA participated in sample collection and sorting. WN, JS, LC,YC and QW conceived the study and revised the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Written informed consent was obtained from the adult patients and the guardians of minor participants. The study protocol was approved by the ethics review boards of relevant institutions, and the use of the anonymized samples was approved by the institutional review board of China Medical University.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing intrests.
Author details
1 Department of Immunology, College of Basic Medical Science, China Medical University, Shenyang 110122, Liaoning, China. 2 Department of Blood Transfusion Medicine, General Hospital of Northern Theater Command, Shenyang 110015, Liaoning, China. 3 Department of Blood Transfusion, Yantaishan Hospital, Yantai 264000, Shandong , China. 4 Central Laboratory, the first hospital of China Medical University, Shenyang 110001, Liaoning, China. 5 Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 6 Global Health Infectious Disease Research (GHIDR) Program, College of Public Health, Tampa, Florida, USA. 7 Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, FL 33612, USA.
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