Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152–625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157–560 aa.) and PvRBP1a-C (606–962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.     

Analysis of polymorphic regions of Plasmodium vivax Duffy binding protein of Korean isolates.

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.     

The association of Duffy binding protein region II polymorphisms and its antigenicity in Plasmodium vivax isolates from Thailand

Plasmodium vivax Duffy binding protein II (DBPII) plays an important role in reticulocyte invasion and is a potential vaccine candidate against vivax malaria. However, polymorphisms in DBPII are a challenge for the successful design of a broadly protective vaccine. In this study, the genetic diversity of DBPII among Thai isolates was analyzed from Plasmodium vivax-infected blood samples and polymorphism characters were defined with the MEGA4 program. Sequence analysis identified 12 variant residues that are common among Thai DBPII haplotypes with variant residues L333F, L424I, W437R and I503K having the highest frequency. Variant residue D384K occurs in combination with either E385K or K386N/Q. Additionally, variant residue L424I occurs in conjunction with W437R in most Thai DBPII alleles and these variants frequently occur in combination with the I503K variant. The polymorphic patterns of Thai isolates were defined into 9 haplotypes (Thai DBL-1, -2, -3, etc.…). Thai DBL-2, -5, -6 haplotypes are the most common DBPII variants in Thai residents. To study the association of these Thai DBPII polymorphisms with antigenic character, the functional inhibition of anti-DBPII monoclonal antibodies against a panel of Thai DBL variants was characterized by an in vitro erythrocyte binding inhibition assay. The functional inhibition of anti-DBPII monoclonal antibodies 3C9, 2D10 and 2C6 against Thai variants was significantly different, suggesting that polymorphisms of Thai DBPII variants alter the antigenic character of the target epitopes. In contrast, anti-DBPII monoclonal antibody 2H2 inhibited all Thai DBPII variants equally well. Our results suggest that the immune efficacy of a DBPII vaccine will depend on the specificity of the anti-DBPII antibodies induced and that it is preferable to optimize responses to conserved epitopes for broadly neutralizing protection against P. vivax.     

Genetic diversity of Plasmodium vivax in a hyperendemic area predominated by Plasmodium falciparum; a preliminary study

Understanding the genetic diversity, extent and distribution of variant forms of Plasmodium vivax parasites is crucial in the development of effective control measures and in Orissa, a hyperendemic state in the eastern part of India, the polymorphic nature of P. vivax isolates is largely lacking. The result of the study analyzing two highly polymorphic single copy genes for P. vivax circumsporozoite protein (pvcs) and P. vivax merozoite surface protein 3α (pvmsp3α) shows that the parasite population is highly heterogenous (33 distinct genotype from 35 isolates) in Orissa. However, the observation of the multiplicity of infection value of 1.34 and high frequency distribution of certain genotype with respect to individual marker (the VK247b allele with a frequency of 0.37; VK210e with 0.25 and VK210c with 0.14) suggests that the parasite population are likely to be under selective pressure and may either be due to preferential production of sporozoites carrying these variants in the available anopheline mosquito species of the state or selection of particular genotypes by host immune pressure. Moreover, although P. vivax in South-East Asia indicates an overall predominance of VK210 which is thought to be the best adapted variant of pvcs repeat type, the almost equal prevalence of both repeat type of pvcs; VK210 and VK247 in the present study is unexpected and needs further study for clarification.     

Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes

Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition.Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.     

The genetic diversity of Plasmodium vivax populations

Little is known of the genetic diversity and population structure of Plasmodium vivax, a debilitating and highly prevalent malaria parasite of humans. This article reviews the known polymorphic genetic markers, summarizes current data on the population structure of this parasite and discusses future prospects for using knowledge of the genetic diversity to improve control measures.     

Plasmodium vivax: karyotype polymorphism of field isolates

Pulse-field gradient electrophoresis (PFG) has been applied to the karyotype analysis of Plasmodium vivax isolates obtained directly from infected patients in Sri Lanka. Detection of separated chromosomes was performed either by ethidium bromide staining of gels or by hybridization with a telomer specific probe. Each of the 15 different isolates examined exhibited a different chromosome migration pattern, indicating that a high level of polymorphism prevailed in wild populations of P. vivax. Chromosome size variation was further confirmed using a P. vivax chromosome-specific probe which also demonstrated that, in each isolate, the parasite population appeared to be homogeneous. These observations were made directly on parasites from infected blood, without the necessity for culture amplification, indicating that PFG can be used on a large scale for the epidemiological analysis of wild parasite populations.     

Age-dependent cellular immune responses to Plasmodium vivax Duffy binding protein in humans

The Plasmodium vivax merozoite Duffy binding protein (DBP) contains a cysteine-rich region II (DBPII) that binds to the Duffy Ag receptor for chemokines on erythrocytes, which is essential for parasite invasion. Cellular immune responses to DBPII have not been reported in P. vivax endemic populations, although they may contribute to partial acquired immunity. To examine host cellular immunity to DBPII and identify major T cell epitopes, PBMCs from 107 individuals (2-68 years old) were examined for cytokine production by ELISPOT and/or ELISA to rDBP and overlapping peptides (displaced by 2 aa spanning a 170-aa region of DBPII corresponding to the critical binding motif to the Duffy Ag receptor for chemokines). In P. vivax-exposed subjects, 60 and 71% generated significant rDBP-induced IFN-gamma and IL-10 production, respectively, 11% stimulated IL-2, and IL-5 and IL-13 were not detected. Children <5 years of age had reduced levels and frequency of rDBP-induced IL-10 and IFN-gamma production compared with partially immune older children and adults (p < 0.01). Five major T cell epitopes were identified. Three of these T cell epitopes contained polymorphic residues present in the population. Peptides synthesized corresponding to these variants induced IFN-gamma and IL-10 production to one variant and little response to the other variant in the same individual. These results demonstrate age-dependent and variant-specific cellular immune responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in endemic populations.     

Targeting the Plasmodium vivax Duffy-binding protein

Plasmodium vivax requires interaction with the Duffy antigen receptor for chemokines (DARC) to enable its invasion of human erythrocytes. Interaction with DARC is mediated by the P. vivax Duffy-binding protein (PvDBP) and is essential for junction formation, which is a key step in the invasion process. The receptor-binding domain of PvDBP maps to a conserved cysteine-rich region, referred to as region II (PvRII). Here, we review data on the interaction of PvRII with DARC and explore the potential of targeting this crucial receptor-ligand interaction to develop new intervention strategies against P. vivax.     

Plasmodium vivax: polymorphism in the merozoite surface protein 1 gene from wild Colombian isolates

The Plasmodium vivax merozoite surface protein-1 (PvMSP-1) has been considered a candidate for a malaria vaccine against erythrocytic stages. PvMSP-1 is immunogenic during natural infections and exhibits antigenic polymorphism. The extent of genetic polymorphism in a region between the so-called interspecies conserved blocks (ICBs) 2 and 4 of the PvMSP-1 was analyzed in 20 isolates taken from patients from two different areas in Colombia. Variation is unevenly distributed along this gene segment among the isolates. Comparative analysis of these sequences led to the definition of five sequence types (ST1 to 5). ST1 to ST4 exhibit a variation pattern associated with sequences present in the Salvador or Belem sequences. However, ST5 has clusters of sequence that have not been previously described. The changes found along the five variants confirm the important role of recombinational and/or gene conversion events in generating allelic diversity.     

Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry.

BACKGROUND: The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two-color flow cytometric erythrocyte binding assay (F-EBA) that has several advantages over traditional erythrocyte binding assays (T-EBAs) used in malaria research. We demonstrate the use of F-EBA using the P. vivax Duffy binding protein region II (PvDBP-RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. METHODS: F-EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP-RII, a fluorescent anti-His tag detection antibody, and thiazole orange before flow cytometric analysis. T-EBAs employing immunoblot detection with an anti-His antibody were performed concomitantly. RESULTS: PvDBP-RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy- erythrocytes, by F-EBAs and T-EBAs. However, F-EBAs exhibited higher sensitivity and better concordance between experiments compared with T-EBAs. CONCLUSIONS: F-EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F-EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T-EBAs in identifying novel erythrocyte binding proteins.     

The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity

A novel class of RNA-binding proteins, Puf, regulates translation and RNA stability by binding to specific sequences in the 3'-untranslated region of target mRNAs. Members of this protein family share a conserved Puf domain consisting of eight 36 amino acid imperfect repeats. Here we report two Puf family member genes, PfPuf1 and PfPuf2, from the human malaria parasite Plasmodium falciparum. Both genes are spliced with four and three introns clustered within or near the Puf domains, respectively. Northern and RT-PCR analysis indicated that both genes were differentially expressed in gametocytes during erythrocytic development of the parasite. Except for similarities in the Puf domain and expression profile, the deduced PfPuf1 and PfPuf2 proteins differ considerably in size and structure. PfPuf1 has 1894 amino acids and a central Puf domain, whereas PfPuf2 is much smaller with a C-terminal Puf domain. The presence of at least two Puf members in other Plasmodium species suggests that these proteins play evolutionarily similar roles during parasite development. Both in vivo studies using the yeast three-hybrid system and in vitro binding assays using the recombinant Puf domain of PfPuf1 expressed in bacteria demonstrated intrinsic binding activity of the PfPuf1 Puf domain to the NRE sequences in the hunchback RNA, the target sequence for Drosophila Pumilio protein. Altogether, these results suggest that PfPufs might function during sexual differentiation and development in Plasmodium through a conserved mechanism of translational regulation of their target mRNAs.     

Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax

The population structure of Plasmodium vivax remains elusive. The markers of choice for large-scale population genetic studies of eukaryotes, short tandem repeats known as microsatellites, have been recently reported to be less polymorphic in P. vivax. Here we investigate the microsatellite diversity and geographic structure in P. vivax, at both local and global levels, using 14 new markers consisting of tri- or tetranucleotide repeats. The local-level analysis, which involved 50 field isolates from Sri Lanka, revealed unexpectedly high diversity (average virtual heterozygosity [H(E)], 0.807) and significant multilocus linkage disequilibrium in this region of low malaria endemicity. Multiple-clone infections occurred in 60% of isolates sampled in 2005. The global-level analysis of field isolates or monkey-adapted strains identified 150 unique haplotypes among 164 parasites from four continents. Individual P. vivax isolates could not be unambiguously assigned to geographic populations. For example, we found relatively low divergence among parasites from Central America, Africa, Southeast Asia and Oceania, but substantial differentiation between parasites from the same continent (South Asia and Southeast Asia) or even from the same country (Brazil). Parasite relapses, which may extend the duration of P. vivax carriage in humans, are suggested to facilitate the spread of strains across continents, breaking down any pre-existing geographic structure.     

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.     

Two new genotypes of Plasmodium vivax circumsporozoite protein found in the Republic of Korea

The gene encoding Plasmodium vivax circumsporozoite protein (PvCSP) exhibits polymorphism in many geographical isolates. The present study was designed to investigate polymorphism in PvCSP gene of P. vivax isolates in Korea. Thirty isolates, obtained from indigenous cases in Yonchon-gun, Kyonggi-do in 1997, were subjected for sequencing and RFLP analysis of the repeat and post-repeat regions of PvCSP gene and two genotypes (SK-A and SK-B) were identified. The genotype of 19 isolates was SK-A and that of 11 isolates was SK-B. Although the number of 12-base repeats present in SK-A was three while two were found in a Chinese strain CH-5, the repeat sequence of SK-A was identical to that of CH-5 except for one base substitution. Compared with known data there was no identical isolates with SK-B, but the sequence of SK-B was similar to that of a North Korean (NK) isolate. These results indicate that two genotypes of PvCSP coexist in the present epidemic area of Korea and the present parasite may originate from East Asia. RFLP would be useful to classify genotypes of P. vivax population instead of gene sequencing.     

Plasmodium vivax: genetic diversity of the apical membrane antigen-1 (AMA-1) in isolates from India

Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that apical membrane antigen-1 (AMA-1) is an effective target for eliciting a protective immune response in humans and other experimental animals. We have investigated the sequence variation in Plasmodium vivax AMA-1 (Pv AMA-1) from different Indian isolates. This is the first study of its kind for the nearly full length Pv AMA-1 from India. Our analysis reveals greater degree of genetic diversity in Pv AMA-1 than reported so far and identifies five novel haplotypes. This is significant to establish the antigenic repertoire of isolates in a malaria endemic country like India.     

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simioovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence.     

Aspartic proteases of Plasmodium vivax are highly conserved in wild isolates

The plasmepsins are the aspartic proteases of malaria parasites. Treatment of aspartic protease inhibitor inhibits hemoglobin hydrolysis and blocks the parasite development in vitro suggesting that these proteases might be exploited their potentials as antimalarial drug targets. In this study, we determined the genetic variations of the aspartic proteases of Plasmodium vivax (PvPMs) of wild isolates. Two plasmepsins (PvPM4 and PvPM5) were cloned and sequenced from 20 P. vivax Korean isolates and two imported isolates. The sequences of the enzymes were highly conserved except a small number of amino acid substitutions did not modify key residues for the function or the structure of the enzymes. The high sequence conservations between the plasmepsins from the isolates support the notion that the enzymes could be reliable targets for new antimalarial chemotherapeutics.     

Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development

The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.