Characterization of merozoite-specific thrombospondin-related anonymous protein (MTRAP) in Plasmodium vivax and P. knowlesi parasites.

Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.

Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein.

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Merozoite surface protein 1 paralog is involved in the human erythrocyte invasion of a zoonotic malaria, Plasmodium knowlesi.

The zoonotic malaria parasite Plasmodium knowlesi is an important public health concern in Southeast Asia. Invasion of host erythrocytes is essential for parasite growth, and thus, understanding the repertoire of parasite proteins that enable this process is vital for identifying vaccine candidates and how some species are able to cause zoonotic infection. Merozoite surface protein 1 (MSP1) is found in all malaria parasite species and is perhaps the most well-studied as a potential vaccine candidate. While MSP1 is encoded by a single gene in P. falciparum, all other human infective species (P. vivax, P. knowlesi, P. ovale, and P. malariae) additionally encode a divergent paralogue known as MSP1P, and little is known about its role or potential functional redundancy with MSP1. We, therefore, studied the function of P. knowlesi merozoite surface protein 1 paralog (PkMSP1P), using both recombinant protein and CRISPR-Cas9 genome editing. The recombinant 19-kDa C-terminus of PkMSP1P (PkMSP1P-19) was shown to bind specifically to human reticulocytes. However, immunoblotting data suggested that PkMSP1P-19-induced antibodies can recognize PkMSP1-19 and vice versa, confounding our ability to separate the properties of these two proteins. Targeted disruption of the pkmsp1p gene profoundly impacts parasite growth, demonstrating for the first time that PkMSP1P is important in in vitro growth of P. knowlesi and likely plays a distinct role from PkMSP1. Importantly, the MSP1P KO also enabled functional characterization of the PkMSP1P-19 antibodies, revealing clear immune cross-reactivity between the two paralogues, highlighting the vital importance of genetic studies in contextualizing recombinant protein studies.

Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) interacts with human CD36, suggesting a novel ligand-receptor interaction for reticulocyte invasion.

BACKGROUND: The Plasmodium vivax merozoite restrictively invades immature erythrocytes, suggesting that its ligand(s) might interact with corresponding receptor(s) that are selectively abundant on reticulocytes to complete the invasion. Finding the ligand‒receptor interaction involved in P. vivax invasion is critical to vivax malaria management; nevertheless, it remains to be unraveled. METHODS: A library of reticulocyte receptors and P. vivax ligands were expressed by a HEK293E mammalian cell expression system and were then used to screen the interaction using enzyme-linked immunosorbent assay (ELISA). A flow cytometry-based erythrocyte binding assay and bio-layer interferometry experiment were further utilized to cellularly and quantitatively identify the ligand‒receptor interaction, respectively. RESULTS: Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) was found to interact with human CD36 using systematic screening. This interaction was specific at a molecular level from in vitro analysis and comparable to that of P. vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) (KD: 37.0 ± 1.4 nM and 7.7 ± 0.5 nM, respectively). Flow cytometry indicated that PvMTRAP preferentially binds to reticulocytes, on which CD36 is selectively present. CONCLUSIONS: Human CD36 is selectively abundant on reticulocytes and is able to interact specifically with PvMTRAP, suggesting that it may function as a ligand and receptor during the invasion of reticulocytes by P. vivax.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

In-depth biological analysis of alteration in Plasmodium knowlesi-infected red blood cells using a noninvasive optical imaging technique.

BACKGROUND: Imaging techniques are commonly used to understand disease mechanisms and their biological features in the microenvironment of the cell. Many studies have added to our understanding of the biology of the malaria parasite Plasmodium knowlesi from functional in vitro and imaging analysis using serial block-face scanning electron microscopy (SEM). However, sample fixation and metal coating during SEM analysis can alter the parasite membrane. METHODS: In this study, we used noninvasive diffraction optical tomography (DOT), also known as holotomography, to explore the morphological, biochemical, and mechanical alterations of each stage of P. knowlesi-infected red blood cells (RBCs). Each stage of the parasite was synchronized using Nycodenz and magnetic-activated cell sorting (MACS) for P. knowlesi and P. falciparum, respectively. Holotomography was applied to measure individual three-dimensional refractive index tomograms without metal coating, fixation, or additional dye agent. RESULTS: Distinct profiles were found on the surface area and hemoglobin content of the two parasites. The surface area of P. knowlesi-infected RBCs showed significant expansion, while P. falciparum-infected RBCs did not show any changes compared to uninfected RBCs. In terms of hemoglobin consumption, P. falciparum tended to consume hemoglobin more than P. knowlesi. The observed profile of P. knowlesi-infected RBCs generally showed similar results to other studies, proving that this technique is unbiased. CONCLUSIONS: The observed profile of the surface area and hemoglobin content of malaria infected-RBCs can potentially be used as a diagnostic parameter to distinguish P. knowlesi and P. falciparum infection. In addition, we showed that holotomography could be used to study each Plasmodium species in greater depth, supporting strategies for the development of diagnostic and treatment strategies for malaria.

Identification of Reticulocyte Binding Domain of Plasmodium ovale curtisi Duffy Binding Protein (PocDBP) Involved in Reticulocyte Invasion.

The Plasmodium ovale curtisi (Poc) prevalence has increased substantially in sub-Saharan African countries as well as regions of Southeast Asia. Poc parasite biology has not been explored much to date; in particular, the invasion mechanism of this malaria parasite remains unclear. In this study, the binding domain of the Duffy binding protein of P. ovale curtisi (PocDBP) was characterized as an important ligand for reticulocyte invasion. The homologous region of the P. vivax Duffy binding protein in PocDBP, named PocDBP-RII herein, was selected, and the recombinant PocDBP-RII protein was expressed in an Escherichia coli system. This was used to analyze reticulocyte binding activity using fluorescence-activated cell sorting and immune serum production in rabbits. The binding specificity was proven by treating reticulocytes with trypsin, chymotrypsin and neuraminidase. The amino acid sequence homology in the N-terminal Cys-rich region was found to be ~ 44% between PvDBP and PocDBP. The reticulocyte binding activity of PocDBP-RII was significantly higher than the erythrocyte binding activity and was concentration dependent. Erythrocyte binding was reduced significantly by chymotrypsin treatment and inhibited by an anti-PocDBP-RII antibody. This finding suggests that PocDBP may be an important ligand in the reticulocyte invasion process of P. ovale curtisi.

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool.

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson's correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.

Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi.

Malaria is caused by multiple different species of protozoan parasites, and interventions in the pre-elimination phase can lead to drastic changes in the proportion of each species causing malaria. In endemic areas, cross-reactivity may play an important role in the protection and blocking transmission. Thus, successful control of one species could lead to an increase in other parasite species. A few studies have reported cross-reactivity producing cross-immunity, but the extent of cross-reactive, particularly between closely related species, is poorly understood. P. vivax and P. knowlesi are particularly closely related species causing malaria infections in SE Asia, and whilst P. vivax cases are in decline, zoonotic P. knowlesi infections are rising in some areas. In this study, the cross-species reactivity and growth inhibition activity of P. vivax blood-stage antigen-specific antibodies against P. knowlesi parasites were investigated. Bioinformatics analysis, immunofluorescence assay, western blotting, protein microarray, and growth inhibition assay were performed to investigate the cross-reactivity. P. vivax blood-stage antigen-specific antibodies recognized the molecules located on the surface or released from apical organelles of P. knowlesi merozoites. Recombinant P. vivax and P. knowlesi proteins were also recognized by P. knowlesi- and P. vivax-infected patient antibodies, respectively. Immunoglobulin G against P. vivax antigens from both immune animals and human malaria patients inhibited the erythrocyte invasion by P. knowlesi. This study demonstrates that there is extensive cross-reactivity between antibodies against P. vivax to P. knowlesi in the blood stage, and these antibodies can potently inhibit in vitro invasion, highlighting the potential cross-protective immunity in endemic areas.

3D morphological and biophysical changes in a single tachyzoite and its infected cells using three-dimensional quantitative phase imaging.

Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis in the human body and commonly infects warm-blooded organisms. Pathophysiology of its diseases is still an interesting issue to be studied since T gondii can infect nearly all nucleated cells. Imaging techniques are crucial for studying its pathophysiology. In T gondii-infected cells structural and biochemical alterations occurred. To study that modification, we use digital holotomography to investigate the structure and biochemical alteration of single tachyzoite and its infected cells in a label-free and quantitative manner. Quantification analysis was done by measuring the refractive index distribution, which provides information about the concentration and dry mass of individual cells. This study showed that holotomography could be effectively used to identify the structural and biochemical alteration in tremendously different cells in supporting pathophysiological research in particular for T gondii-caused diseases.

Surveillance on the Vivax Malaria in Endemic Areas in the Republic of Korea Based on Molecular and Serological Analyses.

Plasmodium vivax reemerged in 1993. It has been sustained for more than 25 years and become one of the important indigenous parasitic diseases in northern and western parts of the Republic of Korea near the demilitarized zone. In particular, relapse is a significant concern for the control of malaria, as short- and long-term incubation periods vary among those infected in Korea. In this study, the prevalence of asymptomatic carriers was examined among residents of high endemic areas of vivax malaria during nonseasonal transmission of mosquitoes. Blood samples from 3 endemic regions in northwestern Korea were evaluated by microscopic examination, rapid diagnostic testing, and nested PCR to identify asymptomatic patients carrying malaria parasites in the community. However, no positive malaria case among residents of endemic areas was detected. Additionally, serological analysis was carried out to measure antibodies against 3 antigenic recombinant proteins of P. vivax, merozoite surface protein 1-19, circumsporozoite surface protein-VK210, and liver-stage antigen (PvLSA-N), by the protein array method. Interestingly, seropositivity of sera between previous exposure and samples without exposure to malaria was significantly higher using the PvLSA-N antigen than the other antigens, suggesting that PvLSA-N can be used as a serological marker to analyze the degree of exposure for malaria transmission in endemic areas. This indicates a very low asymptomatic carrier prevalence during the nonmalaria season in the endemic areas of Korea.

Evaluation of antibody responses to the early transcribed membrane protein family in Plasmodium vivax.

BACKGROUND: Malaria parasites form intracellular membranes that separate the parasite from the internal space of erythrocytes, and membrane proteins from the parasites are exported to the host via the membrane. In our previous study, Plasmodium vivax early transcribed membrane protein (PvETRAMP) 11.2, an intracellular membrane protein that is highly expressed in blood-stage parasites, was characterized as a highly immunogenic protein in P. vivax malaria patients. However, the other PvETRAMP family proteins have not yet been investigated. In this study, PvETRAMPs were expressed and evaluated to determine their immunological profiles. METHODS: The protein structure and amino acid alignment were carried out using bioinformatics analysis software. A total of six PvETRAMP family proteins were successfully expressed and purified using a wheat germ cell free protein expression system and the purified proteins were used for protein microarray and immunization of mice. The localization of the protein was determined with serum against PvETRAMP4. IgG subclasses were assessed from the immunized mice. RESULTS: In silico analysis showed that P. vivax exhibits nine genes encoding the ETRAMP family. The ETRAMP family proteins are relatively small molecules with conserved structural features. A total of 6 recombinant ETRAMP proteins were successfully expressed and purified. The serum positivity of P. vivax malaria patients and healthy individuals was evaluated using a protein microarray method. Among the PvETRAMPs, ETRAMP4 showed the highest positivity rate of 62%, comparable to that of PvETRAMP11.2, which served as the positive control, and a typical export pattern of PvETRAMP4 was observed in the P. vivax parasite. The assessment of IgG subclasses in mice immunized with PvETRAMP4 showed high levels of IgG1 and IgG2b. PvETRAMP family proteins were identified and characterized as serological markers. CONCLUSIONS: The relatively high antibody responses to PvETRAMP4 as well as the specific IgG subclasses observed in immunized mice suggest that the ETRAMP family is immunogenic in pathogens and can be used as a protein marker and for vaccine development.

The persistence of naturally acquired antibodies and memory B cells specific to rhoptry proteins of Plasmodium vivax in patients from areas of low malaria transmission.

BACKGROUND: Rhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. Plasmodium vivax rhoptry proteins have been shown to induce humoral immunity during natural infections. Therefore, these proteins may be potential novel vaccine candidates. However, there is a lack of data on the duration of antibody and memory B cell (MBC) responses. Here, the longitudinal analysis of antibody and MBC responses to the P. vivax rhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence. METHODS: Thirty-nine samples from P. vivax-infected subjects (age 18-60 years) were recruited to explore the frequency and persistence of antibody and MBC responses against rhoptry proteins (PvRALP1-Ecto and PvRhopH2) using both cross-sectional and longitudinal cohort study designs. Antibody levels were determined by ELISA during clinical malaria, and at 3, 9 and 12 months post-infection. The frequency of MBC sub-sets and presence of rhoptry-specific MBCs in subjects 18 months after treatment were detected by flow cytometry and ELISPOT assay. RESULTS: The seroprevalence of antibodies against PvRALP1-Ecto and PvRhopH2 proteins was found to be high during acute infection, with IgG1, IgG2 and IgG3 sub-classes predominant. However, these anti-rhoptry responses were short-lived and significantly decreased at 9 months post-infection. To relate the durability of these antibody responses to MBC persistence at post-infection, 18-month post-infection peripheral blood mononuclear cells (PBMCs) samples were taken to detect rhoptry-specific MBCs and frequency of MBC sub-sets, and correlate with antibody responses. These late post-infection samples revealed that rhoptry-specific MBCs were present in about 70% of total subjects. However, the persistence of specific MBCs was not correlated with antibody responses as the majority of malaria subjects who were positive for PvRALP1-Ecto- or PvRhopH2-specific MBCs were seronegative for the rhoptry antigens. The frequencies of classical MBCs were increased after infection, whereas those of activated and atypical MBCs were decreased, indicating that MBC responses could switch from activated or atypical MBCs to classical MBCs after parasite clearance, and were maintained in blood circulating at post-infection. CONCLUSION: The study showed that rhoptry antigens induced the development and persistence of MBC responses in P. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.

Diversity pattern of Duffy binding protein sequence among Duffy-negatives and Duffy-positives in Sudan.

BACKGROUND: Vivax malaria is a leading public health concern worldwide. Due to the high prevalence of Duffy-negative blood group population, Plasmodium vivax in Africa historically is less attributable and remains a neglected disease. The interaction between Duffy binding protein and its cognate receptor, Duffy antigen receptor for chemokine plays a key role in the invasion of red blood cells and serves as a novel vaccine candidate against P. vivax. However, the polymorphic nature of P. vivax Duffy binding protein (DBP), particularly N-terminal cysteine-rich region (PvDBPII), represents a major obstacle for the successful design of a DBP-based vaccine to enable global protection. In this study, the level of pvdbpII sequence variations, Duffy blood group genotypes, number of haplotypes circulating, and the natural selection at pvdbpII in Sudan isolates were analysed and the implication in terms of DBP-based vaccine design was discussed. METHODS: Forty-two P. vivax-infected blood samples were collected from patients from different areas of Sudan during 2014-2016. For Duffy blood group genotyping, the fragment that indicates GATA-1 transcription factor binding site of the FY gene (- 33T > C) was amplified by PCR and sequenced by direct sequencing. The region II flanking pvdbpII was PCR amplified and sequenced by direct sequencing. The genetic diversity and natural selection of pvdbpII were done using DnaSP ver 5.0 and MEGA ver 5.0 programs. Based on predominant, non-synonymous, single nucleotide polymorphisms (SNPs), prevalence of Sudanese haplotypes was assessed in global isolates. RESULTS: Twenty SNPs (14 non-synonymous and 6 synonymous) were identified in pvdbpII among the 42 Sudan P. vivax isolates. Sequence analysis revealed that 11 different PvDBP haplotypes exist in Sudan P. vivax isolates and the region has evolved under positive selection. Among the identified PvDBP haplotypes five PvDBP haplotypes were shared among Duffy-negative as well as Duffy-positive individuals. The high selective pressure was mainly found on the known B cell epitopes (H3) of pvdbpII. Comparison of Sudanese haplotypes, based on 10 predominant non-synonymous SNPs with 10 malaria-endemic countries, demonstrated that Sudanese haplotypes were prevalent in most endemic countries. CONCLUSION: This is the first pvdbp genetic diversity study from an African country. Sudanese isolates display high haplotype diversity and the gene is under selective pressure. Haplotype analysis indicated that Sudanese haplotypes are a representative sample of the global population. However, studies with a large number of samples are needed. These findings would be valuable for the development of PvDBP-based malaria vaccine.

In vitro invasion inhibition assay using antibodies against Plasmodium knowlesi Duffy binding protein alpha and apical membrane antigen protein 1 in human erythrocyte-adapted P. knowlesi A1-H.1 strain.

BACKGROUND: The rapid process of malaria erythrocyte invasion involves ligand-receptor interactions. Inducing antibodies against specific ligands or receptors that abrogate the invasion process is a key challenge for blood stage vaccine development. However, few candidates were reported and remain to be validated for the discovery of new vaccine candidates in Plasmodium knowlesi. METHODS: In order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals. RESULTS: PkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively. CONCLUSION: These data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.

Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi.

The Plasmodium falciparum apical asparagine (Asn)-rich protein (AARP) is one of malarial proteins, and it has been studied as a candidate of malaria subunit vaccine. Basic characterization of PvAARP has been performed with a focus on its immunogenicity and localization. In this study, we further analyzed the immunogenicity of PvAARP, focusing on the longevity of the antibody response, cross-species immunity and invasion inhibitory activity by using the primate malaria parasite Plasmodium knowlesi. We found that vivax malaria patient sera retained anti-PvAARP antibodies for at least one year without re-infection. Recombinant PvAARP protein was strongly recognized by knowlesi malaria patients. Antibody raised against the P. vivax and P. knowlesi AARP N-termini reacted with the apical side of the P. knowlesi merozoites and inhibited erythrocyte invasion by P. knowlesi in a concentration-dependent manner, thereby suggesting a cross-species nature of anti-PvAARP antibody against PkAARP. These results can be explained by B cell epitopes predicted in conserved surface-exposed regions of the AARP N-terminus in both species. The long-lived anti-PvAARP antibody response, cross-reactivity, and invasion inhibitory activity of anti-PvAARP support a critical role of AARP during the erythrocyte invasion and suggest that PvAARP induces long-lived cross-species protective immunity against P. vivax and P. knowlesi.

Estimation on local transmission of malaria by serological approach under low transmission setting in Myanmar.

BACKGROUND: As the prevalence of the malaria has been decreasing in many endemic countries including Myanmar, malaria elimination in Greater Mekong Region was targeted not later than 2030. The relevance of molecular and serological tools to identify residual transmission remains to be established in this setting. METHODS: One-year cohort study was conducted and sera samples were collected in every 3 months with active and passive case detection for clinical malaria episodes by RDT, microscopy and molecular method. The sera were used to detect the malaria antibody against PfMSP1-19, PvAMA1, PvDBPII and PvMSP1-19 by protein microarray. RESULTS: Among the recruited 1182 participants, there was no RDT positive case for malaria infection although two vivax infections were detected by microscopy in initial collection. Molecular methods detected the asymptomatic cases of 28/1182 (2.37%) in first, 5/894 (0.42%) in second, 12/944 (1.02%) in third, 6/889 (0.51%) in fourth collection, respectively. Seropositivity rates against the PfMSP1-19, PvMSP1-19, PvAMA1 and PvDBPII were 73/270 (27.0%), 85/270 (31.5%), 65/270 (24.1%) and 160/270 (59.3%), respectively. PfMSP1-19 and PvMSP1-19 showed high and stable antigenicity in acute and subacute samples but declining in 1-year history samples. No cross reactivity of PfMSP1-19 and PvMSP1-19 between the two species and higher seropositivity among the asymptomatic carriers were observed. Mapping data indicated serological surveillance can detect the geographical pattern of malaria infection under low transmission setting. CONCLUSIONS: These findings support that PfMSP1-19 and PvMSP1-19 are suggested for serosurveillance of the malaria especially in low transmission setting for further necessary actions have to be carried out to eliminate the malaria.

Immunogenicity of the Plasmodium vivax merozoite surface protein 1 paralog in the induction of naturally acquired antibody and memory B cell responses.

BACKGROUND: The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P-19) is a glycosylphosphatidylinositol (GPI)-anchored blood-stage protein that is expressed on the merozoite surface. It is proposed as a blood-stage vaccine candidate against P. vivax because of its ability to induce immune responses upon natural P. vivax exposure and in immunized animals. This study aimed to demonstrate the presence of inhibitory antibodies and memory B cell responses to the PvMSP1P-19 antigen during acute P. vivax infection and after recovery from infection. METHODS: To evaluate the antibody responses to PvMSP1P-19 during and after recovery from P. vivax infection, heparinized blood was collected from P. vivax-infected patients and recovered subjects to detect the total IgG response. The seropositive samples were defined into high and low responders, according to their optical density (OD) values obtained from ELISA. High responders were the subjects who had OD values above the OD of antisera from non-exposed controls plus 4× standard deviations, whereas low responders were the subjects who had OD values less than OD of antisera from non-exposed controls plus 4× standard deviations. The plasma from high and low responders were taken for testing the inhibitory activity against PvMSP1P-19-erythrocyte binding by in vitro EBIA. The sustainability of PvMSP1P-19-specific memory B cell responses after recovery from infection was analysed by ELISPOT. RESULTS: The anti-PvMSP1P-19 antibody levels were significantly higher in acutely infected P. vivax patients compared to healthy controls (P <  0.0001). Monitoring of the anti-PvMSP1P-19 antibody titre showed that the antibody was maintained for up to 9 months after recovery. Almost all high-responder groups strongly inhibited PvMSP1P-19 binding to erythrocytes, whereas no inhibition was shown in most low-responder samples. Interestingly, the inhibitory activity of the antibodies in some individuals from high-responder samples were stable for at least 12 months. The longevity of the antibody response was associated with the presence of PvMSP1P-19-specific memory B cells at 9 months after recovery from infection. CONCLUSIONS: The PvMSP1P-19 antigen has immunogenicity during the induction of the antibody response, in which both the levels and inhibitory activity are maintained after the patient recovered from P. vivax infection. The maintenance of the antibody response was associated with the response of PvMSP1P-19-specific memory B cells. Therefore, the PvMSP1P-19 antigen should also be considered as a reliable vaccine candidate to develop a blood-stage vaccine against P. vivax.

Molecular surveillance of artemisinin resistance falciparum malaria among migrant goldmine workers in Myanmar.

BACKGROUND: Artemisinin resistance has been reported in Greater Mekong Sub-region countries, including Myanmar. After discovery of artemisinin resistance marker (K13), molecular surveillance on artemisinin resistance in endemic regions have been conducted. As the migrant population represents a high percentage of malaria cases, molecular surveillance of artemisinin resistance among migrant workers is of great concern. METHODS: A cross-sectional survey was conducted in Shwegyin Township, where migrants work in the goldmines. Blood samples were collected from uncomplicated Plasmodium falciparum-infected migrant workers by active and passive cases screening with rapid diagnostic testing (RDT) and microscopy. Amplification and sequence analysis of artemisinin resistance molecular markers, such as k13, pfarps10, pffd, pfmdr2, pfmrp1, pfrad5, and pfcnbp, were carried out and pfmdr1 copy number analysis was conducted by real-time PCR. RESULTS: Among the 100 falciparum-infected patients, most were male (90%), of working age (20-40 years) with median parasite density of 11,166 parasites/µL (range 270-110,472 parasites/µL). Artemisinin resistance molecular marker, k13 mutations were detected in (21/100, 21.0%) in which composed of a validated marker, C580Y (9/21, 42.9%) and candidate markers such as P574L (5/21, 23.8%), P667T (5/21, 23.8%) and M476I (2/21, 9.5%). Underlying genetic markers predisposing to become k13 mutants were found as V127M of pfarps10 (41/100, 41.0%), D153Y of pffd (64/100, 64.0%), T484I of pfmdr2 (58/100, 58.0%) and F1390I of pfmrp1 (24/100, 24.0%). The pfmdr1 copy number analysis revealed six copy numbers (1/100, 1.0%), three (2/100, 2.0%), two (8/100, 8.0%) and only one copy number (89/100, 89.0%). Only one sample showed both k13 mutation (P667T) and multiple copy number of pfmdr1. CONCLUSIONS: High mutant rate of artemisinin resistance markers and relatively high pfmdr1 copy number among isolates collected from migrant goldmine workers alert the importance of containment measures among this target population. Clinical and molecular surveillance of artemisinin resistance among migrants should be scaled up.