Identifying and characterizing a member of the RhopH1/Clag family in Plasmodium vivax

Plasmodium vivax malaria caused is a public health problem that produces very high morbidity worldwide. During invasion of red blood cells the parasite requires the intervention of high molecular weight complex rhoptry proteins that are also essential for cytoadherence. PfClag9, a member of the RhopH multigene family, has been identified as being critical during Plasmodium falciparum infection. This study describes identifying and characterizing the pfclag9 ortholog in P. vivax (hereinafter named pvclag7). The pvclag7 gene is transcribed at the end of the intraerythrocytic cycle and is recognized by sera from humans who have been infected by P. vivax. PvClag7 subcellular localization has been also determined and, similar to what occurs with PfClag9, it co-localize with other proteins from the Rhoptry high molecular weight complex.     

Plasmodium vivax malaria: parasite biology defines potential targets for vaccine development

Although progress in the development of an antimalarial vaccine has been mostly obtained through the study of P. falciparum, significant advances have recently been made in the study of P. vivax, the other major human malarial parasite. Antigens which have been shown to be important in P. falciparum have been characterized and in some cases cloned in P. vivax. Other studies have examined some of the more specific biological characteristics of P. vivax. Among these are studies on components present in caveolae-vesicle complexes of the infected erythrocyte, on the occurrence of delayed hepatic development leading to relapse, or on the Duffy erythrocyte antigen as a key receptor for parasite invasion. Although progress has been made in the short-term in vitro maintenance of P. vivax, the inability to maintain the parasite in continuous culture led to the investigation of wild parasite populations in patients; occurrence of extensive antigenic and karyotype polymorphism was detected in this way, as was a double-blocking and enhancing activity of human antibodies on parasite development in the vector. The association of monoclonal antibodies with DNA recombinant technology allowed the characterization of a number of P. vivax antigens to be made. Among these, an antigen shared between sexual and asexual stages was shown to constitute a target for transmission-blocking immunity. The cloning of an antigen involved in transmission-blocking immunity, along with that of the surface antigen of the sporozite (CSP) and of a major surface antigen of the invasive merozoite (PV200) constitutes a significant step towards the development of a multivalent recombinant vaccine against P. vivax.     

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.     

Lineage-specific positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium vivax and related simian malaria parasites

Background

The 200 kDa merozoite surface protein 1 (MSP-1) of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors). It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity.

Results

We performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1) from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species-specifically on msp1.

Conclusions

The present results indicate that the msp1 locus of P. vivax and related parasite species has lineage-specific unique evolutionary history with positive selection. P. vivax and related simian malaria parasites offer an interesting system toward understanding host species-dependent adaptive evolution of immune-target surface antigen genes such as msp1.     

Progress in understanding the phylogeny of the Plasmodium vivax lineage

There has been some controversy about the evolutionary origin of Plasmodium vivax, particularly whether it is of Asian or African origin. Recently, a new malaria species which closely related to ape P. vivax was found in chimpanzees, in addition, the host switches of P. vivax from ape to human was confirmed. These findings support the African origin of P. vivax. Previous phylogenetic analyses have shown the position of P. vivax within the Asian primate malaria parasite clade. This suggested an Asian origin of P. vivax. Recent analyses using massive gene data, however, positioned P. vivax after the branching of the African Old World monkey parasite P. gonderi, and before the branching of the common ancestor of Asian primate malaria parasites. This position is consistent with an African origin of P. vivax. We here review the history of phylogenetic analyses on P. vivax, validate previous analyses, and finally present a definitive analysis using currently available data that indicate a tree in which P. vivax is positioned at the base of the Asian primate malaria parasite clade, and thus that is consistent with an African origin of P. vivax.     

Molecular Evidence of Plasmodium vivax Mono and Mixed Malaria Parasite Infections in Duffy-Negative Native Cameroonians

The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans.     

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.     

Babesia argentina, Plasmodium vivax and P. falciparum: antigenic cross-reactions

An indirect fluorescent antibody test was used to analyze the antigenic relationships between Babesia argentina, a parasite of cattle, and two human malaria parasites, Plasmodium falciparum and Plasmodium vivax. Elevated antibody titers to P. falciparum were found in cattle infected with B. argentina. Some persons infected with P. falciparum or P. vivax were found to produce antibodies to B. argentina. Explanations for the occurrence of these cross reactions are considered.     

Plasmodium vivax – How hidden reservoirs hinder global malaria elimination

Plasmodium vivax is the most geographically widespread human malaria parasite. Global malaria efforts have been less successful at reducing the burden of P. vivax compared to P. falciparum, owing to the unique biology and related treatment complexity of P. vivax. As a result, P. vivax is now the dominant malaria parasite throughout the Asia-Pacific and South America causing up to 14 million clinical cases every year and is considered a major obstacle to malaria elimination. Key features circumventing existing malaria control tools are the transmissibility of asymptomatic, low-density circulating infections and reservoirs of persistent dormant liver stages (hypnozoites) that are undetectable but reactivate to cause relapsing infections and sustain transmission. In this review we summarise the new knowledge shaping our understanding of the global epidemiology of P. vivax infections, highlighting the challenges for elimination and the tools that will be required achieve this.     

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.     

The Plasmodium vivax Merozoite Surface Protein 3β Sequence Reveals Contrasting Parasite Populations in Southern and Northwestern Thailand

Background

Malaria control efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Thailand.

Methodology

We employed the P. vivax merozoite surface protein 3β (PvMSP3β) as a molecular marker for characterizing P. vivax populations based on the extensive diversity of this gene in Southeast Asian parasite populations. To examine two parasite populations with different transmission levels in Thailand, we obtained 45 P. vivax isolates from Tak Province, northwestern Thailand, where the annual parasite incidence (API) was more than 2%, and 28 isolates from Yala and Narathiwat Provinces, southern Thailand, where the API was less than 0.02%. We sequenced the PvMSP3β gene and examined its genetic diversity and molecular evolution between the parasite populations.

Principal Findings

Of 58 isolates containing single PvMSP3β alleles, 31 sequence types were identified. The overall haplotype diversity was 0.77±0.06 and nucleotide diversity 0.0877±0.0054. The northwestern vivax malaria population exhibited extensive haplotype diversity (HD) of PvMSP3β (HD = 1.0). In contrast, the southern parasite population displayed a single PvMSP3β allele (HD = 0), suggesting a clonal population expansion. This result revealed that the extent of allelic diversity in P. vivax populations in Thailand varies among endemic areas.

Conclusion

Malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into account for the implementation of P. vivax control measures such as drug policy and vaccine development.     

Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein

Background

Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes.

Methods and Findings

Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%.

Conclusions

These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.     

Increased reticulocyte count from cord blood samples using hypotonic lysis

Human reticulocytes are one of the fundamental components needed to study the in vitro invasion processes of the human malaria parasite Plasmodium vivax. Additionally examinations of reticulocytes and their binding proteins are difficult in areas of the world that do not have access to advanced equipment or stem cell lines. These issues are particularly relevant to malaria vaccine candidate studies that are directed against surface proteins that the parasites use to gain entry into erythrocytes. Described here is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. Exposure of cord blood to hypotonic saline (0.2%) for 5 min selectively lyses the non-reticulocytes resulting in an average 3.6-fold increase in reticulocyte count. Our studies show that this enrichment process does not damage the hemoglobin of the remaining erythrocytes which are still capable of supporting Plasmodium falciparum invasion and growth. This economical and rapid method of enrichment could facilitate studies of in vitro laboratory culturing of other malaria parasite species which preferentially invade reticulocytes such as P. vivax.     

The Role of Reactive Oxygen Species in Anopheles aquasalis Response to Plasmodium vivax Infection

Malaria affects millions of people worldwide and hundreds of thousands of people each year in Brazil. The mosquito Anopheles aquasalis is an important vector of Plasmodium vivax, the main human malaria parasite in the Americas. Reactive oxygen species (ROS) have been shown to have a role in insect innate immune responses as a potent pathogen-killing agent. We investigated the mechanisms of free radicals modulation after A. aquasalis infection with P. vivax. ROS metabolism was evaluated in the vector by studying expression and activity of three key detoxification enzymes, one catalase and two superoxide dismutases (SOD3A and SOD3B). Also, the involvement of free radicals in the mosquito immunity was measured by silencing the catalase gene followed by infection of A. aquasalis with P. vivax. Catalase, SOD3A and SOD3B expression in whole A. aquasalis were at the same levels of controls at 24 h and upregulated 36 h after ingestion of blood containing P. vivax. However, in the insect isolated midgut, the mRNA for these enzymes was not regulated by P. vivax infection, while catalase activity was reduced 24 h after the infectious meal. RNAi-mediated silencing of catalase reduced enzyme activity in the midgut, resulted in increased P. vivax infection and prevalence, and decreased bacterial load in the mosquito midgut. Our findings suggest that the interactions between A. aquasalis and P. vivax do not follow the model of ROS-induced parasite killing. It appears that P. vivax manipulates the mosquito detoxification system in order to allow its own development. This can be an indirect effect of fewer competitive bacteria present in the mosquito midgut caused by the increase of ROS after catalase silencing. These findings provide novel information on unique aspects of the main malaria parasite in the Americas interaction with one of its natural vectors.     

Erythrocyte Binding Activity Displayed by a Selective Group of Plasmodium vivax Tryptophan Rich Antigens Is Inhibited by Patients’ Antibodies

Plasmodium vivax is a very common but non-cultivable malaria parasite affecting large human population in tropical world. To develop therapeutic reagents for this malaria, the parasite molecules involved in host-parasite interaction need to be investigated as they form effective vaccine or drug targets. We have investigated here the erythrocyte binding activity of a group of 15 different Plasmodium vivax tryptophan rich antigens (PvTRAgs). Only six of them, named PvTRAg, PvTRAg38, PvTRAg33.5, PvTRAg35.2 PvTRAg69.4 and PvATRAg74, showed binding to host erythrocytes. That the PvTRAgs binding to host erythrocytes was specific was evident from the competitive inhibition and saturation kinetics results. The erythrocyte receptors for these six PvTRAgs were resistant to trypsin and neuraminidase. These receptors were also chymotrypsin resistant except the receptors for PvTRAg38 and PvATRAg74 which were partially sensitive to this enzyme. The cross-competition studies showed that the chymotrypsin resistant RBC receptor for each of these two proteins was different. Altogether, there seems to be three RBC receptors for these six PvTRAgs and each PvTRAg has two RBC receptors. Both RBC receptors for PvTRAg, PvTRAg69.4, PvTRAg33.5, and PvTRAg35.2 were common to all these four proteins. These four PvTRAgs also shared one of their RBC receptors with PvTRAg38 as well as with PvATRAg74. The erythrocyte binding activity of these six PvTRAgs was inhibited by the respective rabbit polyclonal antibodies as well as by the natural antibodies produced by the P. vivax exposed individuals. It is concluded that only selective few PvTRAgs show erythrocyte binding activity involving different receptor molecules which can be blocked by the natural antibodies. Further studies on these receptor and ligands may lead to the development of therapeutic reagents for P. vivax malaria.     

Prevalence of Drug Resistance-Associated Gene Mutations in Plasmodium vivax in Central China

Resistance of Plasmodium spp. to anti-malarial drugs is the primary obstacle in the fight against malaria, and molecular markers for the drug resistance have been applied as an adjunct in the surveillance of the resistance. In this study, we investigated the prevalence of mutations in pvmdr1, pvcrt-o, pvdhfr, and pvdhps genes in temperate-zone P. vivax parasites from central China. A total of 26 isolates were selected, including 8 which were previously shown to have a lower susceptibility to chloroquine in vitro. For pvmdr1, pvcrt-o, and pvdhps genes, no resistance-conferring mutations were discovered. However, a highly prevalent (69.2%), single-point mutation (S117N) was found in pvdhfr gene. In addition, tandem repeat polymorphisms existed in pvdhfr and pvdhps genes, which warranted further studies in relation to the parasite resistance to antifolate drugs. The study further suggests that P. vivax populations in central China may still be relatively susceptible to chloroquine and sulfadoxine-pyrimethamine.     

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.     

Structural analyses of the malaria parasite aminoacyl‐tRNA synthetases provide new avenues for antimalarial drug discovery

Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl‐tRNA synthetases (aaRSs) have been explored as anti‐bacterial and anti‐fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure‐based knowledge of the most advanced three aaRSs—lysyl‐ (KRS), prolyl‐ (PRS), and phenylalanyl‐ (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.