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.     

Population genetic structure of the Plasmodium vivax circumsporozoite protein (Pvcsp) in Sri Lanka

Molecular methods elucidate evolutionary and ecological processes in parasites, where interaction between hosts and parasites enlighten the evolution of parasite lifestyles and host defenses. Population genetics of Plasmodium vivax parasites accurately describe transmission dynamics of the parasites and evaluation of malaria control measures. As a first generation vaccine candidate against malaria, the Circumsporozoite Protein (CSP) has demonstrated significant potential in P. falciparum. Extensive polymorphism hinders the development of a potent malaria vaccine. Hence, the genetic diversity of Pvcsp was investigated for the first time in 60 Sri Lankan clinical isolates by obtaining the nucleotide sequence of the central repeat (CR) domain and examining the polymorphism of the peptide repeat motifs (PRMs), the genetic diversity indices and phylogenetic relationships. PCR amplicons determined size polymorphism of 610, 700 and 710 bp in Pvcsp of Sri Lanka where all amino acid sequences obtained were of the VK210 variant, consisting variable repeats of 4 different PRMs. The two most abundant PRMs of the CR domain, GDRADGQPA and GDRAAGQPA consisted ~ 2-4 repeats, while GNRAAGQPA was unique to the island. Though, different nucleotide sequences termed repeat allotypes (RATs) were observed for each PRM, these were synonymous contributing to a less polymorphic CR domain. The genetic diversity of Pvcsp in Sri Lanka was due to the number of repetitive peptide repeat motifs, point mutations, and intragenic recombination. The 19 amino acid haplotypes defined were exclusive to Sri Lanka, whereas the 194 Pvcsp sequences of global isolates generated 57 more distinct a.a. haplotypes of the VK210 variant. Strikingly, the CR domain of both VK210 and VK247 variants was under purifying selection interpreting the scarcity of CSP non-synonymous polymorphisms. Insights to the distribution of RATs in the CR region with geographic clustering of the P. vivax VK210 variant were revealed. The cladogram reiterated this unique geographic clustering of local (VK210) and global isolates (VK210 and VK247), which was further validated by the elevated fixation index values of the VK210 variant.     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

Polymorphisms in Plasmodium vivax Circumsporozoite Protein (CSP) Influence Parasite Burden and Cytokine Balance in a Pre-Amazon Endemic Area from Brazil

Mechanisms involved in severe P. vivax malaria remain unclear. Parasite polymorphisms, parasite load and host cytokine profile may influence the course of infection. In this study, we investigated the influence of circumsporozoite protein (CSP) polymorphisms on parasite load and cytokine profile in patients with vivax malaria. A cross-sectional study was carried out in three cities: São Luís, Cedral and Buriticupu, Maranhão state, Brazil, areas of high prevalence of P. vivax. Interleukin (IL)-2, IL-4, IL-10, IL-6, IL-17, tumor necrosis factor alpha (TNF-α, interferon gamma (IFN-γ and transforming growth factor beta (TGF-β were quantified in blood plasma of patients and in supernatants from peripheral blood mononuclear cell (PBMC) cultures. Furthermore, the levels of cytokines and parasite load were correlated with VK210, VK247 and P. vivax-like CSP variants. Patients infected with P. vivax showed increased IL-10 and IL-6 levels, which correlated with the parasite load, however, in multiple comparisons, only IL-10 kept this association. A regulatory cytokine profile prevailed in plasma, while an inflammatory profile prevailed in PBMC culture supernatants and these patterns were related to CSP polymorphisms. VK247 infected patients showed higher parasitaemia and IL-6 concentrations, which were not associated to IL-10 anti-inflammatory effect. By contrast, in VK210 patients, these two cytokines showed a strong positive correlation and the parasite load was lower. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In conclusion, we propose that CSP polymorphisms is associated to the increase of non-regulated inflammatory immune responses, which in turn may be associated with the outcome of infection.     

Prevalence and level of antibodies to the circumsporozoite protein of human malaria parasites in five states of the Amazon region of Brazil

The aim of this study was to determine the prevalence of malaria infection and antibodies against the repetitive epitopes of the circumsporozoite (CS) proteins of Plasmodium falciparum, P. malariae, P. vivax VK210, P. vivax VK247, and P. vivax-like in individuals living in the states of Rond?nia, Pará, Mato Grosso, Amazonas, and Acre. Active malaria transmission was occurring in all studied sites, except in Acre. P. falciparum was the predominant species in Pará and Rond?nia and P. vivax in Mato Grosso. Infection by P. malariae was low but this Plasmodium species was detected in Rond?nia (3.5%), Mato Grosso (2.5%), and Pará (0.8%). High prevalence and levels of serological reactivity against the CS repeat peptides of P. falciparum were detected in Rond?nia (93%) and Pará (85%). Sera containing antibodies against the CS repeat of P. malariae occurred more frequently in Rond?nia (79%), Pará (76%), and Amazonas (68%). Antibodies against the repeat epitope of the standard CS protein of P. vivax VK210, P. vivax VK247, and P. vivax-like were more frequent in Rond?nia, Pará, and Mato Grosso. The high frequency of reactions to P. malariae in most of the areas suggests that the infection by this Plasmodium species has been underestimated in Brazil.     

Comparison of immunological responses to the various types circumsporozoite proteins of Plasmodium vivax in malaria patients of Korea

We compared the seroreactivities against four synthetic peptide antigens (VK210, VK247, Korean type 1, and type 2) and a full length recombinant circumsporozoite protein (CSP) antigen of Plasmodium vivax (P. vivax ) in samples of sixty-three tertian malaria patients in Korea. Among the various CSP antigens, the full-length recombinant CSP showed the highest reactivity in malaria-exposed groups (85.7%, 54/63). No significant difference was found in the percentage of malaria patients with antibodies among four peptides examined, except a full-length recombinant CSP. Absorbance values from the peptide-based ELISA showed high correlations (r > 0.9, P < 0.05) at significant values. Five sera without the immunoaffinity against peptides were reactive towards the full-length recombinant CSP in ELISA. Sera, which were not reactive to a full length recombinant CSP antigen, were not recognized by any of peptide based ELISA. These data suggested that peptide structures included in Korean isolates, GNGAGGQAA, and VK247 peptides had immune reactivity and recognition epitopes. Among the antigens, GNGAGGQAA was less recognized by patients exposed to Korean strains of P. vivax in comparison to the VK210 structures.     

Susceptibility of Anopheles aquasalis and an. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F3 generation) and An. darlingi (F1 generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.     

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.     

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.     

Apical membrane antigen-1 (AMA-1) gene sequences of re-emerging Plasmodium vivax in South Korea

Plasmodium vivax malaria re-emerged in South Korea in 1993, and epidemics continue since then. We examined genetic variation in the region encompassing the apical membrane antigen-1 (PvAMA-1) of the parasites by DNA sequencing of the 22 re-emerging P. vivax isolates. The genotype of the PvAMA-1, which was based on sequence data previously reported for the polymorphic regions, showed that two haplotypes were present at one polymorphic site. Compared with reported data, the two types, SKOR type I and type II, were similar to Chinese CH-10A and CH-05A isolates, respectively. Thus, the present study showed that two genotypes of AMA-1 genes coexist in the re-emerging Korean P. vivax.     

Positive diversifying selection on Plasmodium vivax RON2 protein

Plasmodium rhoptry neck protein 2 (RON2), which is released from the neck portion of the merozoite rhoptries and interacts with the microneme protein Apical Membrane Antigen 1 (AMA1), plays a crucial role in erythrocyte invasion. In this study, we sequenced the Plasmodium vivax RON2 gene from 19 P. vivax isolates collected in central China in order to establish whether this protein is under positive diversifying selection, which may occur as a result of protective host immune pressure?. In comparison with the P. vivax Sal-1 reference line, we found 10 amino acid substitutions dispersed throughout the open reading frame as well as indels caused by polymorphism in a repeat unit (21-23 repeats of (Q/E/K/N/H)(G/D)G(H/L/Y/P)G) in the second tandem repeat region located at amino acid positions 541-650. A McDonald-Kreitman test with RON2 sequences from the primate malaria parasite Plasmodium knowlesi, detected significant departure from neutrality in the PvRON2 3' region (nucleotide positions 2668-6609). These results suggest that the PvRON2 gene has evolved under positive diversifying selection.     

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.     

Widespread occurrence of antibodies against circumsporozoite protein and against blood forms of Plasmodium vivax, P. falciparum and P. malariae in Brazilian wild monkeys

BACKGROUND: A survey of malaria antibodies was carried out over 7 years and a total of 777 serum samples from wild monkeys were collected in three distinct ecological areas of Brazil where autochthonous malaria has been reported: the 'Cerrado' (similar to savanna), the Atlantic Forest and the Atlantic Semideciduous Forest. METHODS: We carried out enzyme-linked immunosorbent assay to investigate the presence of IgG antibodies against peptides of the circumsporozoite protein (CSP) repeat region of 'classic'Plasmodium vivax, P. vivax VK247, human P. vivax-like/P. simiovale, P. brasilianum/P. malariae and P. falciparum. We also carried out immunofluorescence assay with asexual forms of P. vivax, P. malariae and P. falciparum. RESULTS: The high prevalence of antibodies against CSP in all areas indicates that the monkeys had intense contact with sporozoites from infected anophelines. The immune response against asexual forms of Plasmodium in the monkeys from the Atlantic Forest indicates the development of the infection. CONCLUSIONS: We discuss the possibility of monkeys being malaria reservoirs in non-endemic areas.     

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.     

Plasmodium vivax dhfr mutations among isolates from malarious areas of Iran

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.     

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.     

Plasmodium vivax CS peptides display conformational preferences for folded forms in solution.

Spectroscopic techniques have been used to study the conformations of several synthetic peptides with sequences corresponding to the repeat regions of the circumsporozoite proteins of Plasmodium vivax, variants vk-210 and vk-247. As has previously been shown for P. falciparum, turn-like folded conformations are observed, in rapid dynamic equilibrium with extended-chain forms. These results are consistent with the known similarity of the structural, biosynthetic and immunological properties of the circumsporozoite proteins of different plasmodial species. Additionally, the observation of folded conformers provides a rationale for the effectiveness of these peptides as immunogens and potential vaccines.     

Determination of the Plasmodium vivax schizont stage proteome

With the genome of the malaria parasite Plasmodium vivax sequenced, it is important to determine the proteomes of the parasite in order to assist efforts in antigen and drug target discovery. Since a method for continuous culture of P. vivax parasite is not available, we tried to study the proteome of the erythrocytic stages using fresh parasite isolates from patients. In schizont-enriched samples, 316 proteins were confidently identified by tandem mass spectrometry. Almost 50% of the identified proteins were hypothetical, while other major categories include proteins with binding function, protein fate, protein synthesis, metabolism and cellular transport. To identify proteins that are recognized by host humoral immunity, parasite proteins were separated by two-dimensional gel electrophoresis and screened by Western blot using an immune serum from a P. vivax patient. Mass spectrometry analysis of protein spots recognized by the serum identified four potential antigens including PV24. The recombinant protein PV24 was recognized by antibodies from vivax malaria patients even during the convalescent period, indicating that PV24 could elicit long-lasting antibody responses in P. vivax patients.     

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.     

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.