Vivax Merozoite Surface Protein Research Articles

Genetic Diversity and Natural Selection of Plasmodium vivax Merozoite Surface Protein 8 in Global Populations

Plasmodium vivax Merozoite Surface Protein 8 (PvMSP8) is a promising candidate target for the development of multi-component vaccines. Therefore, determining the genetic variation pattern of Pvmsp8 is essential in providing a reference for the rational design of the P. vivax malaria vaccines. This study delves into the genetic characteristics of the Pvmsp8 gene, specifically focusing on samples from the China-Myanmar border (CMB) region, and contrasts these findings with broader global patterns. The study uncovers that Pvmsp8 exhibits a notable level of conservation across different populations, with limited polymorphisms and relatively low nucleotide diversity (0.00023–0.00120). This conservation contrasts starkly with the high polymorphisms found in other P. vivax antigens such as Pvmsp1. A total of 25 haplotypes and 14 amino acid mutation sites were identified in the global populations, and all mutation sites were confined to non-functional regions. The study also notes that most CMB Pvmsp8 haplotypes are shared among Burmese, Cambodian, Thai, and Vietnamese populations, indicating less geographical variance, but differ notably from those found in Pacific island regions or the Panama. The findings underscore the importance of considering regional genetic diversity in P. vivax when developing targeted malaria vaccines. Non departure from neutral evolution were found by Tajima's D test, however, statistically significant differences were observed between the kn/ks rates. The study's findings are crucial in understanding the evolution and population structure of the Pvmsp8 gene, particularly during regional malaria elimination efforts. The highly conserved nature of Pvmsp8, combined with the lack of mutations in its functional domain, presents it as a promising candidate for developing a broad and effective P. vivax vaccine. This research thus lays a foundation for the rational development of multivalent malaria vaccines targeting this genetically stable antigen.

Anti-Plasmodium vivax merozoite surface protein 3 ϒ (PvMSP3 ϒ) antibodies upon natural infection

Merozoite surface protein 3 of Plasmodium vivax (PvMSP3) contains a repertoire of protein members with unique sequence organization. While the biological functions of these proteins await elucidation, PvMSP3 has been suggested to be potential vaccine targets. To date, studies on natural immune responses to this protein family have been confined to two members, PvMSP3α and PvMSP3β. This study analyzed natural IgG antibody responses to PvMSP3γ recombinant proteins derived from two variants: one containing insert blocks (CT1230nF) and the other without insert domain (NR25nF). The former variant was also expressed as two subfragment proteins: one encompassing variable domain I and insert block A (CT1230N) and the other spanning from insert block B to conserved block III (CT1230C). Serum samples were obtained from 246 symptomatic vivax malaria patients in Tak (n = 50) and Ubon Ratchathani (n = 196) Provinces. In total, 176 (71.5%) patients could mount antibodies to at least one recombinant PvMSP3γ antigen. IgG antibodies directed against antigens CT1230nF, CT1230N, CT1230C and NR25nF occurred in 96.6%, 61.4%, 71.6% and 68.2% of samples, respectively, suggesting the widespread occurrence of B-cell epitopes across PvMSP3γ. The rates of seropositivity seemed to correlate with the number of previous malaria episodes. Isotype analysis of anti-PvMSP3γ antibodies has shown predominant cytophilic subclass responses, accounting for 75.4–81.7% for IgG1 and 63.6–77.5% for IgG3. Comparing with previous studies in the same cohort, the numbers of serum samples reactive to antigens derived from P. vivax merozoite surface protein 9 (PvMSP9) and thrombospondin-related anonymous protein (PvTRAP) were higher than those to PvMSP3γ, being 92.7% and 87.0% versus 71.5%, respectively. Three (1.22%) serum samples were nonresponsive to all these malarial proteins. Nevertheless, the relevance of naturally acquired antibodies to PvMSP3γ in host protection requires further studies.

Genetic diversity and molecular evolution of Plasmodium vivax Duffy Binding Protein and Merozoite Surface Protein-1 in northwestern Thailand.

The local diversity and population structure of malaria parasites vary across different regions of the world, reflecting variations in transmission intensity, host immunity, and vector species. This study aimed to use amplicon sequencing to investigate the genotypic patterns and population structure of P. vivax isolates from a highly endemic province of Thailand in recent years. Amplicon deep sequencing was performed on 70 samples for the 42-kDa region of pvmsp1 and domain II of pvdbp. Unique haplotypes were identified and a network constructed to illustrate genetic relatedness in northwestern Thailand. Based on this dataset of 70 samples collected between 2015 and 2021, 16 and 40 unique haplotypes were identified in pvdbpII and pvmsp142kDa, respectively. Nucleotide diversity was higher in pvmsp142kDa than in pvdbpII (π=0.027 and 0.012), as was haplotype diversity (Hd=0.962 and 0.849). pvmsp142kDa also showed a higher recombination rate and higher levels of genetic differentiation (Fst) in northwestern Thailand versus other regions (0.2761-0.4881). These data together suggested that the genetic diversity of P. vivax in northwestern Thailand at these two studied loci evolved under a balancing selection, most likely host immunity. The lower genetic diversity of pvdbpII may reflect its stronger functional constrain. In addition, despite the balancing selection, a decrease in genetic diversity was observed. Hd of pvdbpII decreased from 0.874 in 2015-2016 to 0.778 in 2018-2021; π of pvmsp142kDa decreased from 0.030 to 0.022 over the same period. Thus, the control activities must have had a strong impact on the parasite population size. The findings from this study provide an understanding of P. vivax population structure and the evolutionary force on vaccine candidates. They also established a new baseline for tracking future changes in P. vivax diversity in the most malarious area of Thailand.

Distinct Allelic Diversity of Plasmodium vivax Merozoite Surface Protein 3-Alpha (PvMSP-3α) Gene in Thailand Using PCR-RFLP.

Considering the importance of merozoite surface proteins (MSPs) as vaccine candidates, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax merozoite surface protein 3-alpha (PvMSP-3α) in Thailand. To analyze genetic diversity, 118 blood samples containing P. vivax were collected from four malaria-endemic areas in western and southern Thailand. The DNA was extracted and amplified for the PvMSP-3α gene using nested PCR. The PCR products were genotyped by PCR-RFLP with Hha I and Alu I restriction enzymes. The combination patterns of Hha I and Alu I RFLP were used to identify allelic variants. Genetic evaluation and phylogenic analysis were performed on 13 sequences, including 10 sequences from our study and 3 sequences from GenBank. The results revealed three major types of PvMSP-3α, 91.5% allelic type A (∼1.8 kb), 5.1% allelic type B (∼1.5 kb), and 3.4% allelic type C (∼1.2 kb), were detected based on PCR product size with different frequencies. Among all PvMSP-3α, 19 allelic subtypes with Hha I RFLP patterns were distinguished and 6 allelic subtypes with Alu I RFLP patterns were identified. Of these samples, 73 (61%) and 42 (35.6%) samples were defined as monoallelic subtype infection by Hha I and Alu I PCR-RFLP, respectively, whereas 77 (65.3%) samples were determined to be mixed-allelic subtype infection by the combination patterns of Hha I and Alu I RFLP. These results strongly indicate that PvMSP-3α gene is highly polymorphic, particularly in blood samples collected from the Thai-Myanmar border area (the western part of Thailand). The combination patterns of Hha I and Alu I RFLP of the PvMSP-3α gene could be considered for use as molecular epidemiologic markers for genotyping P. vivax isolates in Thailand.

Genetic diversity of Plasmodium vivax populations from the China–Myanmar border identified by genotyping merozoite surface protein markers

BackgroundParasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China–Myanmar border.MethodsWe collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3β, by PCR-restriction fragment length polymorphism (PCR–RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations.ResultsPCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3β among the 370 and 378 samples, respectively. PCR–RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3β with Pst I. PCR–RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3β. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion.ConclusionsMigrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR–RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-3 Alpha and Beta from Diverse Geographic Areas of Thailand.

Malaria is parasitic disease cause by Plasmodium infection. In Thailand, coinfections of Plasmodium vivax and Plasmodium falciparum are commonly found. P. vivax infection has been increasing in the past decade. The objective of this study was to investigate the genetic diversity patterns of P. vivax merozoite surface protein 3 (PvMSP-3) genes in a total of 450 isolates collected from Thai-neighboring border areas during two different periods (2009-2014 and 2015-2016) using polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Three major types of PvMSP-3α (A, B, and C) and PvMSP-3β (A, B, and C) were detected based on PCR product size. In total, 45 and 23 alleles of PvMSP-3α and 41 and 30 alleles of PvMSP-3β with different frequencies of samples were distinguished from the first and second period, respectively. Our results strongly indicate genetic diversity patterns of PvMSP-3 in isolates from the second period, especially in samples from the Thai-Myanmar border area. These two polymorphic genes could be used as molecular epidemiologic markers for genotyping P. vivax isolate in Thailand.

Surveillance of Plasmodium vivax transmission using serological models in the border areas of China\u2013Myanmar

BackgroundTo understand the Plasmodium vivax malaria transmission intensity and to assess the effectiveness of prevention and control measures taken along the China–Myanmar border, a catalytic model was used to calculate the seroconversion rate, an important indicator of malaria transmission intensity with high sensitivity, which is particularly useful in areas of low transmission.MethodsFive counties in Yunnan Province bordering Myanmar were selected as survey sites, and subjects were obtained in each county by stratified random sampling in 2013–2014. Fingerstick blood was collected from each subject and tested for antibodies to P. vivax Merozoite Surface Protein 1-19 (PvMSP1-19) using indirect ELISA. A catalytic conversion model was used to assess the transmission intensity of P. vivax malaria based on the maximum likelihood of generating a community seroconversion rate.ResultsA total of 3064 valid blood samples were collected. Antibody levels were positively correlated with age. The seroconversion rate (SCR) values for each village were Luoping (0.0054), Jingqiao (0.0061), Longpen (0.0087), Eluo (0.0079), Banwang (0.0042) and Banbie (0.0046), respectively.ConclusionOverall, the intensity of P. vivax malaria transmission in the border areas of Yunnan Province is low and not entirely consistent across counties. Consecutive serological surveys are needed to provide a sensitive evaluation of transmission dynamics and can help to confirm areas where infection is no longer present.

Microfluidic device based on electrodeposited Nanoporous Gold/Carbon Nanotubes for Plasmodium vivax detection

A portable microfluidic electrochemical immunosensor for Plasmodium vivax antibodies determination was developed. A gold microelectrode placed inside the central channel of the microfluidic device was used as immobilization platform for a specific fragment (19-kDa) derived from the P. vivax merozoite surface protein 1, known as PvMSP119. The gold microelectrode surface was modified by using a dynamic hydrogen bubble template (DHBT) method in the presence of multiwalled carbon nanotubes. The synthesized nanocomposite presented exceptional properties, like high specific surface area, remarkable biocompatibility, and excellent electrochemical activity. The material was characterized by scanning electron microscopy, energy dispersive spectrometry, x-ray diffraction, and cyclic voltammetry. The antibodies (anti-PvMSP119) present in the sample bind to the PvMSP119 immobilized on the microelectrode surface, which is then labeled with an anti-IgG antibody marked with horseradish peroxidase (HRP-anti-IgG). Finally, the substrate solution (H2O2 + catechol) is added, and the enzymatic product (quinone) is reduced on the NPAu electrode at +0.2 V (vs. Ag/AgCl). The current obtained is directly proportional to the anti-PvMSP119 concentration in the sample. The detection limit of the microfluidic electrochemical immunosensor was 0.6 ng mL−1, much lower compared to the ELISA detection limit of 15 ng mL−1. This is the first microfluidic electrochemical immunosensor device suitable for point-of-care determination of anti-PvMSP119 in human serum samples.

Temporal Changes in the Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-1 in Myanmar.

Despite a significant decline in the incidence of malaria in Myanmar recently, malaria is still an important public health concern in the country. Although Plasmodium falciparum is associated with the highest incidence of malaria in Myanmar, the proportion of P. vivax cases has shown a gradual increase in recent years. The genetic diversity of P. vivax merozoite surface protein-1 block 5-6 (pvmsp-1 ICB 5-6) in the P. vivax population of Myanmar was analyzed to obtain a comprehensive insight into its genetic heterogeneity and evolutionary history. High levels of genetic diversity of pvmsp-1 ICB 5-6 were identified in the P. vivax isolates collected from Myanmar between 2013 and 2015. Thirty-nine distinct haplotypes of pvmsp-1 ICB 5-6 (13 for Sal I type, 20 for recombinant type, and 6 for Belem type) were found at the amino acid level. Comparative analyses of the genetic diversity of pvmsp-1 ICB 5-6 sequences in the recent (2013–2015) and the past (2004) P. vivax populations in Myanmar revealed genetic expansion of the pvmsp-1 ICB 5-6 in recent years, albeit with a declined incidence. The recent increase in the genetic heterogeneity of Myanmar pvmsp-1 ICB 5-6 is attributed to a combination of factors, including accumulated mutations and recombination. These results suggest that the size of the P. vivax population in Myanmar is sufficient to enable the generation and maintenance of genetic diversity, warranting continuous molecular surveillance of genetic variation in Myanmar P. vivax.

PvMSP8 as a Novel Plasmodium vivax Malaria Sero-Marker for the Peruvian Amazon

The measurement of recent malaria exposure can support malaria control efforts. This study evaluated serological responses to an in-house Plasmodium vivax Merozoite Surface Protein 8 (PvMSP8) expressed in a Baculovirus system as sero-marker of recent exposure to P. vivax (Pv) in the Peruvian Amazon. In a first evaluation, IgGs against PvMSP8 and PvMSP10 proteins were measured by Luminex in a cohort of 422 Amazonian individuals with known history of Pv exposure (monthly data of infection status by qPCR and/or microscopy over five months). Both serological responses were able to discriminate between exposed and non-exposed individuals in a good manner, with slightly higher performance of anti-PvMSP10 IgGs (area under the curve AUC = 0.78 [95% CI = 0.72–0.83]) than anti-PvMSP8 IgGs (AUC = 0.72 [95% CI = 0.67–0.78]) (p = 0.01). In a second evaluation, the analysis by ELISA of 1251 plasma samples, collected during a population-based cross-sectional survey, confirmed the good performance of anti-PvMSP8 IgGs for discriminating between individuals with Pv infection at the time of survey and/or with antecedent of Pv in the past month (AUC = 0.79 [95% CI = 0.74–0.83]). Anti-PvMSP8 IgG antibodies can be considered as a good biomarker of recent Pv exposure in low-moderate transmission settings of the Peruvian Amazon.

Immunoglobulin G responses to variant forms of Plasmodium vivax merozoite surface protein 9 upon natural infection in Thailand

Merozoite surface protein 9 (MSP9) constitutes a ligand complex involved in erythrocyte invasion by malarial merozoites and is a promising vaccine target. Plasmodium vivax MSP9 (PvMSP9) is immunogenic upon natural malaria exposure. To address whether sequence diversity in PvMSP9 among field isolates could affect natural antibody responses, the recombinant proteins representing two variants each for the N- and the C-terminal domains of PvMSP-9 were used as antigens to assess antibody reactivity among 246 P. vivax-infected patients’ sera from Tak and Ubon Ratchathani Provinces in Thailand. Results revealed that the seropositivity rates of IgG antibodies to the N-terminal antigens were higher than those to the C-terminal antigens (87.80% vs. 67.48%). Most seropositive sera were reactive to both variants, suggesting the presence of common epitopes. Variant-specific antibodies to the N- and the C-terminal antigens were detected in 15.85% and 16.70% of serum samples, respectively. These seropositivity rates were not significant difference between provinces. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies exhibited positive trends towards increasing malaria episodes. The IgG isotype responses to the N- and the C-terminal antigens were mainly IgG1 and IgG3. The profile of IgG responses may have implications for development of PvMSP9-based vaccine.

Two 20-Residue-Long Peptides Derived from Plasmodium vivax Merozoite Surface Protein 10 EGF-Like Domains Are Involved in Binding to Human Reticulocytes.

Plasmodium parasites’ invasion of their target cells is a complex, multi-step process involving many protein-protein interactions. Little is known about how complex the interaction with target cells is in Plasmodium vivax and few surface molecules related to reticulocytes’ adhesion have been described to date. Natural selection, functional and structural analysis were carried out on the previously described vaccine candidate P. vivax merozoite surface protein 10 (PvMSP10) for evaluating its role during initial contact with target cells. It has been shown here that the recombinant carboxyl terminal region (rPvMSP10-C) bound to adult human reticulocytes but not to normocytes, as validated by two different protein-cell interaction assays. Particularly interesting was the fact that two 20-residue-long regions (388DKEECRCRANYMPDDSVDYF407 and 415KDCSKENGNCDVNAECSIDK434) were able to inhibit rPvMSP10-C binding to reticulocytes and rosette formation using enriched target cells. These peptides were derived from PvMSP10 epidermal growth factor (EGF)-like domains (precisely, from a well-defined electrostatic zone) and consisted of regions having the potential of being B- or T-cell epitopes. These findings provide evidence, for the first time, about the fragments governing PvMSP10 binding to its target cells, thus highlighting the importance of studying them for inclusion in a P. vivax antimalarial vaccine.

A Chimeric Plasmodium vivax Merozoite Surface Protein Antibody Recognizes and Blocks Erythrocytic P. cynomolgi Berok Merozoites In Vitro.

Research on erythrocytic Plasmodium vivax merozoite antigens is critical for identifying potential vaccine candidates in reducing P. vivax disease. However, many P. vivax studies are constrained by its inability to undergo long-term culture in vitro Conserved across all Plasmodium spp., merozoite surface proteins are essential for invasion into erythrocytes and highly expressed on erythrocytic merozoites, thus making it an ideal vaccine candidate. In clinical trials, the P. vivax merozoite surface protein 1 (PvMSP1-19) vaccine candidate alone has shown to have limited immunogenicity in patients; hence, we incorporate the highly conserved and immunogenic C terminus of both P. vivax merozoite surface protein 8 (PvMSP8) and PvMSP1-19 to develop a multicomponent chimeric protein rPvMSP8+1 for immunization of mice. The resulted chimeric rPvMSP8+1 antibody was shown to recognize native protein MSP8 and MSP1-19 of mature P. vivax schizonts. In the immunized mice, an elevated antibody response was observed in the rPvMSP8+1-immunized group compared to that immunized with single-antigen components. In addition, we examined the growth inhibition of these antibodies against Plasmodium cynomolgi (Berok strain) parasites, which is phylogenetically close to P. vivax and sustains long-term culture in vitro Similarly, the chimeric anti-rPvMSP8+1 antibodies recognize P. cynomolgi MSP8 and MSP1-19 on mature schizonts and showed strong inhibition in vitro via growth inhibition assay. This study provides support for a new multiantigen-based paradigm rPvMSP8+1 to explore potential chimeric vaccine candidates against P. vivax malaria using sister species P. cynomolgi.

Genetic diversity of Merozoite surface protein 1-42 (MSP1-42) fragment of Plasmodium vivax from Indonesian isolates: Rationale implementation of candidate MSP1 vaccine.

Morbidity and mortality related to malaria in Indonesia are attributed to both Plasmodium falciparum and P. vivax parasites. In addition to vaccines for P. falciparum, vaccines against P. vivax are urgently needed for the prevention of the disease. An extensively studied antigen is the carboxyl-terminus of the 42kDa region of P. vivax merozoite surface protein-1 (PvMSP1-42). The design of a vaccine based on this antigen requires an understanding of the extent of polymorphism. However, there is no information on the genetic diversity of the antigen in Indonesia. This study aimed to profile the diversity of PvMSP1-42 and its two subdomains (PvMSP1-33 and PvMSP1-19) among Indonesian P. vivax isolates. A total of 52 P. vivax-infected blood samples were collected from patients in two different endemic areas in Indonesia: Banjarmasin (Kalimantan) and Sumba Timur (Nusa Tenggara Timur). The polymorphic characteristics and natural selection of PvMSP1-42 were analyzed using the DnaSP, MEGA, and Structure software. Thirty distinct haplotypes of PvMSP1-42 were identified. They displayed amino acid changes compared to the reference PVP01 sequence. Most of the mutations were concentrated in the 33kDa fragment. PvMSP1-42 of the Indonesian isolates appeared to be under positive selection. Recombination may also play a role in the resulting genetic diversity of PvMSP1. In conclusion, PvMSP1-42 of Indonesian isolates displayed allelic polymorphisms caused by mutation, recombination, and positive selection. These results will aid the understanding of the P. vivax population in Indonesia and to develop a PvMSP1 based vaccine against P. vivax.

Structural diversity, natural selection and intragenic recombination in the Plasmodium vivax merozoite surface protein 9 locus in Thailand

The merozoite surface protein 9 (MSP9) of malarial parasite forms co-ligand complex with the 19 kDa fragment of merozoite surface protein 1 (MSP1) prior to erythrocyte invasion. Interruption of this process could hamper subsequent asexual erythrocytic development of malaria parasites; therefore, these proteins are considered potential vaccine candidates. In Plasmodium vivax, MSP9 (PvMSP9) contains both conserved and polymorphic repetitive domains that were immunogenic upon natural malaria exposure and conferred protection in vaccination studies in animal models. To investigate the extent of sequence diversity at this locus, 104 P. vivax isolates from 4 major malaria endemic areas of Thailand were analyzed. Results revealed that pvmsp9 contained 3 repeat domains (R1–R3) flanked by conserved domains. Repeat domains exhibit extensive sequence and length variation, in which 14, 39 and 16 haplotypes for domains R1–R3, respectively, circulated in this country. Sequence diversity in pvmsp9 among P. vivax isolates from each endemic area displayed population structure. The extent of sequence diversity in pvmsp9 isolates from the provinces of Tak, Chanthaburi, Ubon Ratchathani and Prachuap Khiri Khan in northwestern, eastern, northeastern and southwestern areas, respectively, was almost comparable and was remarkably higher than that from Yala/Narathiwat population in southern Thailand. Evidence for intragenic recombination in this locus was observed within each P. vivax population except among isolates from Yala and Narathiwat. Synonymous nucleotide diversity significantly exceeded nonsynonymous nucleotide diversity in domains R2 and R3, indicating purifying selection. However, micro-scale signatures of positive and negative selections occurred in both conserved and repeat domains, implying two opposing forces, probably from functional or structural constraint and host immune pressure, could have influenced diversity at this locus. The immunodominant T and B cell epitopes so far identified were invariant or highly conserved across isolates. Further analysis of global isolates is warranted for vaccine design based on this protein.

Insights into the molecular diversity of Plasmodium vivax merozoite surface protein-3\u03b3 (pvmsp3\u03b3), a polymorphic member in the msp3 multi-gene family

Plasmodium vivax merozoite surface protein 3 (PvMSP3) is encoded by a multi-gene family. Of these, PvMSP3α, PvMSP3β and PvMSP3γ, are considered to be vaccine targets. Despite comprehensive analyses of PvMSP3α and PvMSP3β, little is known about structural and sequence diversity in PvMSP3γ. Analysis of 118 complete pvmsp3γ sequences from diverse endemic areas of Thailand and 9 reported sequences has shown 86 distinct haplotypes. Based on variation in insert domains, pvmsp3γ can be classified into 3 types, i.e. Belem, Salvador I and NR520. Imperfect nucleotide repeats were found in six regions of the gene; none encoded tandem amino acid repeats. Predicted coiled-coil heptad repeats were abundant in the protein and displayed variation in length and location. Interspersed phase shifts occurred in the heptad arrays that may have an impact on protein structure. Polymorphism in pvmsp3γ seems to be generated by intragenic recombination and driven by natural selection. Most P. vivax isolates in Thailand exhibit population structure, suggesting limited gene flow across endemic areas. Phylogenetic analysis has suggested that insert domains could have been subsequently acquired during the evolution of pvmsp3γ. Sequence and structural diversity of PvMSP3γ may complicate vaccine design due to alteration in predicted immunogenic epitopes among variants.

Screening for malaria antigen and anti-malarial IgG antibody in forcibly-displaced Myanmar nationals: Cox\u2019s Bazar district, Bangladesh, 2018

BackgroundSeveral refugee settlements in Bangladesh have provided housing and medical care for the forcibly-displaced Myanmar nationals (FDMN, also known as Rohingya) population. The identification of malaria infection status in the refugee settlements is useful in treating infected persons and in developing malaria prevention recommendations. Assays for Plasmodium antigens and human IgG against Plasmodium parasites can be used as indicators to determine malaria infection status and exposure.MethodsDried blood spot (DBS) samples (N = 1239) from a household survey performed April–May 2018 in three settlements in Cox’s Bazar district, Bangladesh were utilized for a sample population of children from ages 1–14 years of age. The samples were tested using a bead-based multiplex antigen assay for presence of the pan-Plasmodium antigen aldolase as well as Plasmodium falciparum histidine rich protein 2 (HRP2). A bead-based multiplex assay was also used to measure human IgG antibody response to P. falciparum, Plasmodium malariae, and Plasmodium vivax merozoite surface protein 1 antigen (MSP1) isoforms, and P. falciparum antigens LSA1, CSP, and GLURP-R0.ResultsThere were no detectable Plasmodium antigens in any samples, suggesting no active malaria parasite infections in the tested children. IgG seroprevalence was highest to P. vivax (3.1%), but this was not significantly different from the percentages of children antibody responses to P. falciparum (2.1%) and P. malariae (1.8%). The likelihood of an anti-Plasmodium IgG response increased with age for all three malaria species. Evidence of exposure to any malaria species was highest for children residing 8–10 months in the settlements, and was lower for children arriving before and after this period of time.ConclusionsAbsence of Plasmodium antigen in this population provides evidence that children in these three Bangladeshi refugee settlements did not have malaria at time of sampling. Higher rates of anti-malarial IgG carriage from children who were leaving Myanmar during the malaria high-transmission season indicate these migrant populations were likely at increased risk of malaria exposure during their transit.

Induction and Kinetics of Complement-Fixing Antibodies Against Plasmodium vivax Merozoite Surface Protein 3α and Relationship With Immunoglobulin G Subclasses and Immunoglobulin M.

Complement-fixing antibodies are important mediators of protection against Plasmodium falciparum malaria. However, complement-fixing antibodies remain uncharacterized for Plasmodium vivax malaria. P. vivax merozoite surface protein 3α (PvMSP3α) is a target of acquired immunity and a potential vaccine candidate. Plasma from children and adults with P. vivax malaria in Sabah, Malaysia, were collected during acute infection, 7 and 28 days after drug treatment. Complement-fixing antibodies and immunoglobulin M and G (IgM and IgG), targeting 3 distinctive regions of PvMSP3α, were measured by means of enzyme-linked immunosorbent assay. The seroprevalence of complement-fixing antibodies was highest against the PvMSP3α central region (77.6%). IgG1, IgG3, and IgM were significantly correlated with C1q fixation, and both purified IgG and IgM were capable of mediating C1q fixation to PvMSP3α. Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults. Levels of functional antibodies increased after acute infection through 7 days after treatment but rapidly waned by day 28. Our study demonstrates that PvMSP3α antibodies acquired during P. vivax infection can mediate complement fixation and shows the important influence of age in shaping these specific antibody responses. Further studies are warranted to understand the role of these functional antibodies in protective immunity against P. vivax malaria.

A Multi-Stage Plasmodium vivax Malaria Vaccine Candidate Able to Induce Long-Lived Antibody Responses Against Blood Stage Parasites and Robust Transmission-Blocking Activity.

Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4+ T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.

Identification and characterization of Pv50, a novel Plasmodium vivax merozoite surface protein

BackgroundPlasmodium vivax contains approximately 5400 coding genes, more than 40% of which code for hypothetical proteins that have not been functionally characterized. In a previous preliminary screening using pooled serum samples, numerous hypothetical proteins were selected from among those that were highly transcribed in the schizont-stage of parasites, and highly antigenic P. vivax candidates including hypothetical proteins were identified. However, their immunological and functional activities in P. vivax remain unclear. From these candidates, we investigated a P. vivax 50-kDa protein (Pv50, PVX_087140) containing a highly conserved signal peptide that shows high transcription levels in blood-stage parasites.ResultsRecombinant Pv50 was expressed in a cell-free expression system and used for IgG prevalence analysis of patients with vivax malaria and healthy individuals. Immune responses were analyzed in immunized mice and mouse antibodies were used to detect the subcellular localization of the protein in blood-stage parasites by immunofluorescence assay. A protein array method was used to evaluate protein-protein interactions to predict protein functional activities during the invasion of parasites into erythrocytes. Recombinant Pv50 showed IgG prevalence in patient samples with a sensitivity of 42.9% and specificity of 93.8% compared to that in healthy individuals. The non-cytophilic antibodies IgG1 and IgG3 were the major components involved in the antibody response in Pv50-immunized mice. Pv50 localized on the surface of merozoites and a specific interaction between Pv50 and PvMSP1 was detected, suggesting that Pv50-PvMSP1 forms a heterodimeric complex in P. vivax.ConclusionsIncreased immune responses caused by native P. vivax parasites were detected, confirming its immunogenic effects. This study provides a method for detecting new malaria antigens, and Pv50 may be a vivax malaria vaccine candidate with PvMSP1.