Merozoite Surface Protein Research Articles

Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody

Vaccine trials and cohort studies in Plasmodium falciparum endemic areas indicate that naturally-acquired and vaccine-induced antibodies to merozoite surface protein 2 (MSP2) are associated with resistance to malaria. These data indicate that PfMSP2 has significant potential as a component of a multi-antigen malaria vaccine. To overcome challenges encountered with subunit malaria vaccines, we established that the use of highly immunogenic rPfMSP8 as a carrier protein for leading vaccine candidates rPfMSP119 and rPfs25 facilitated antigen production, minimized antigenic competition and enhanced induction of functional antibodies. We applied this strategy to optimize a rPfMSP2 (3D7)-based subunit vaccine by producing unfused rPfMSP2 or chimeric rPfMSP2/8 in Escherichia coli. rPfMSP2 formed fibrils, which induced splenocyte proliferation in an antigen receptor-independent, TLR2-dependent manner. However, fusion to rPfMSP8 prevented rPfMSP2 amyloid-like fibril formation. Immunization of rabbits elicited high-titer anti-PfMSP2 antibodies that recognized rPfMSP2 of the 3D7 and FC27 alleles, as well as native PfMSP2. Competition assays revealed a difference in the specificity of antibodies induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. Rabbit anti-PfMSP2/8 was superior to rPfMSP2-elicited antibody at opsonizing P. falciparum merozoites for phagocytosis. These data establish rPfMSP8 as an effective carrier for a PfMSP2-based subunit malaria vaccine.

Clinical expression and antigenic profiles of a Plasmodium vivax vaccine candidate: merozoite surface protein 7 (PvMSP-7)

BackgroundVivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates.MethodsMerozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs.ResultsTen patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients.ConclusionsThese results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.

Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60–70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.

Genetic dissociation of three antigenic genes in Plasmodium ovale curtisi and Plasmodium ovale wallikeri

Plasmodium ovale curtisi and Plasmodium ovale wallikeri are two sympatric human malaria species prevalent in Africa, Asia and Oceania. The reported prevalence of both P. ovale spp. was relatively low compared to other malaria species, but more sensitive molecular detection techniques have shown that asymptomatic low-density infections are more common than previously thought. Whole genome sequencing of both P. ovale spp. revealed genetic dissociation between P. ovale curtisi and P. ovale wallikeri suggesting a species barrier. In this study we further evaluate such a barrier by assessing polymorphisms in the genes of three vaccine candidate surface protein: circumsporozoite protein/ thrombospondin-related anonymous-related protein (ctrp), circumsporozoite surface protein (csp) and merozoite surface protein 1 (msp1). The complete coding sequence of ctrp and csp, and a partial fragment of msp1 were isolated from 25 P. ovale isolates and compared to previously reported reference sequences. A low level of nucleotide diversity (Pi = 0.02–0.10) was observed in all three genes. Various sizes of tandem repeats were observed in all ctrp, csp and msp1 genes. Both tandem repeat unit and nucleotide polymorphism in all three genes exhibited clear dimorphism between P. ovale curtisi and P. ovale wallikeri, supporting evidence of non-recombination between these two species.

The acquisition of long-lived memory B cell responses to merozoite surface protein-8 in individuals with Plasmodium vivax infection

BackgroundThe ability of a malaria antigen to induce effective, long-lasting immune responses is important for the development of a protective malaria vaccine. Plasmodium vivax merozoite surface protein-8 (PvMSP8) has been shown to be immunogenic in natural P. vivax infections and produces both cell-mediated and antibody-mediated immunity. Thus, PvMSP8 has been proposed as a vaccine candidate following fusion with other merozoite antigens in blood stage vaccine design. Here, the long-term responses of antibodies and memory B cells (MBCs) specific to PvMSP8 in individuals were monitored in a longitudinal cohort study.MethodsBoth cross-sectional surveys and cohort studies were utilized to explore the persistence of antibody and MBC responses to PvMSP8. Antibody titers were detected in individuals with acute disease and those who recovered from an infection for 4 years. The dominant peptide epitope of PvMSP8 recognized by naturally acquired antibodies was examined to observe the durability of the post-infection antibody response. PvMSP8-specific MBCs were also in subjects 4 years post-infection using an enzyme-linked immunospot assay.ResultsThe prevalence of antibodies to PvMSP8 was high during and after infection. The antibody levels in individual responders were monitored for up to 12 months post-infection and showed that most patients maintained their seropositive response. Interestingly, the anti-PvMSP8 antibody responses stably persisted in some patients who had recovered from an infection for 4 years. Positive PvMSP8-specific MBCs were also detected at 4 years post-infection. However, analysis in these individuals showed no correlation with the presence or titer of circulating antibody.ConclusionPvMSP8 had the ability to induce a long-term humoral immune response. The antibodies and MBCs specific for this antigen developed and persisted in subjects who acquired a natural P. vivax infection. Inclusion of the PvMSP8 antigen in blood stage vaccine design should be considered.

Reduced Transplacental Transfer of Antimalarial Antibodies in Kenyan HIV-Exposed Uninfected Infants.

BackgroundAltered neonatal immune responses may contribute to the increased morbidity observed in HIV-exposed but uninfected (HEU) infants compared with HIV-unexposed uninfected (HUU) infants. We sought to examine the effects of prenatal HIV and malaria exposure on maternal and neonatal plasma cytokine profiles and transplacental antibody transfer.MethodsForty-nine HIV+ and 50 HIV- women and their HIV-uninfected neonate pairs from Kenya were assessed. All HIV+ mothers received combination antiretroviral therapy. Maternal plasma and cord blood plasma samples at delivery were tested for 12 cytokines, total IgG, and IgG specific to 4 vaccine antigens and 14 Plasmodium falciparum antigens.ResultsHIV+ mothers had lower levels of all 12 plasma cytokines at delivery compared with HIV- mothers, but there were no differences between HEU and HUU neonates. There were no differences in the cord-to-maternal ratios (CMRs) of vaccine-specific IgG between HIV+/HEU and HIV-/HUU maternal–neonate pairs. HIV+/HEU maternal–neonate pairs had significantly lower CMRs for 3 antimalarial IgGs—merozoite surface protein 9, circumsporozoite protein, and erythrocyte binding antigen 181—which remained statistically significant after adjustment for malaria in pregnancy.ConclusionsIn a cohort of optimally treated HIV-infected pregnant women, maternal HIV infection was associated with reduced transplacental transfer of antimalarial antibodies.

Molecular surveillance of pfhrp2 and pfhrp3 genes deletion in Plasmodium falciparum isolates and the implications for rapid diagnostic tests in Nigeria

Prompt diagnosis and appropriate treatment of malaria remain the hallmark for reducing malaria-related mortality in high transmission areas. Plasmodium falciparum histidine-rich protein2 (PfHRP2) based rapid diagnostic tests (RDT) play a vital role in prompt and accurate malaria diagnosis. However, pfhrp2 gene deletion threatens the RDT test sensitivity. This study reports the presence of pfhrp2 and pfhrp3 genes deletion among parasite isolates in Nigeria. Febrile children were screened using histidine-rich protein (HRP2) specific RDT (SD-Bioline RDT) and microscopy for P. falciparum infections. All RDT negative samples were re-evaluated by polymerase chain reaction (PCR). The presence of parasite in RDT false negative cases and randomly selected RDT positive cases were validated using PCRs targeting glutamate-rich protein (glurp) and merozoite surface proteins (msp-1 and msp-2). Thereafter, exon 2 of pfhrp2 and pfhrp3 were amplified, and Sanger sequenced. A total of 511 febrile children were enrolled out of which 309 (61%) were positive by RDT. The presence of pfhrp2 and pfhrp3 genes were analyzed in 66 PCR positive samples comprising of 31 RDT false negative and 35 RDT true positive randomly selected samples. The pfhrp2 and pfhrp3 genes failed to amplify in 17% (11/66) and 6% (4/66) samples, respectively. Seven of the eleven samples had only pfhrp2 deletion while four had both pfhrp2 and pfhrp3 deletions. The absence of the pfhrp2 gene may be responsible for the seven RDT false negative cases observed. Three RDT positive cases lacked pfhrp2 whereas pfhrp3 was absent in only four RDT false negative cases. The pfhrp2 and pfhrp3 amino acid repeat sequences were highly diverse. The P. falciparum isolates lacking pfhrp2 and pfhrp3 genes may be circulating and contributing to RDT false negativity in Nigeria. More studies in larger population and seasonally defined cases will be needed to determine the extent of pfhrp2/3 genes deletion in different geographical areas of Nigeria.

Contribution of Functional Antimalarial Immunity to Measures of Parasite Clearance in Therapeutic Efficacy Studies of Artemisinin Derivatives.

BackgroundAntibodies to the blood stages of malaria parasites enhance parasite clearance and antimalarial efficacy. The antibody subclass and functions that contribute to parasite clearance during antimalarial treatment and their relationship to malaria transmission intensity have not been characterized.MethodsLevels of immunoglobulin G (IgG) subclasses and C1q fixation in response to Plasmodium falciparum merozoite antigens (erythrocyte-binding antigen [EBA] 175RIII-V, merozoite surface protein 2 [MSP-2], and MSP-142) and opsonic phagocytosis of merozoites were measured in a multinational trial assessing the efficacy of artesunate therapy across 11 Southeast Asian sites. Regression analyses assessed the effects of antibody seropositivity on the parasite clearance half-life (PC½), having a PC½ of ≥5 hours, and having parasitemia 3 days after treatment.ResultsIgG3, followed by IgG1, was the predominant IgG subclass detected (seroprevalence range, 5%–35% for IgG1 and 27%–41% for IgG3), varied across study sites, and was lowest in study sites with the lowest transmission intensity and slowest mean PC½. IgG3, C1q fixation, and opsonic-phagocytosis seropositivity were associated with a faster PC½ (range of the mean reduction in PC½, 0.47–1.16 hours; P range, .001–.03) and a reduced odds of having a PC½ of ≥5 hours and having parasitemia 3 days after treatment.ConclusionsThe prevalence of IgG3, complement-fixing antibodies, and merozoite phagocytosis vary according to transmission intensity, are associated with faster parasite clearance, and may be sensitive surrogates of an augmented clearance capacity of infected erythrocytes. Determining the functional immune mechanisms associated with parasite clearance will improve characterization of artemisinin resistance.

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.

Plasmodium knowlesi clinical isolates from Malaysia show extensive diversity and strong differential selection pressure at the merozoite surface protein 7D (MSP7D)

BackgroundThe high proportion of human cases due to the simian malaria parasite Plasmodium knowlesi in Malaysia is a cause of concern, as they can be severe and even fatal. Merozoite surface protein 7 (MSP7) is a multigene family which forms a non-covalent complex with MSP-1 prior to receptor-ligand recognition in Plasmodium falciparum and thus an important antigen for vaccine development. However, no study has been done in any of the ortholog family members in P. knowlesi from clinical samples. This study investigates the level of polymorphism, haplotypes, and natural selection acting at the pkmsp-7D gene in clinical samples from Malaysia.MethodsThirty-six full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) obtained from clinical isolates of Malaysia, which were orthologous to pvmsp7H (PVX_082680) were downloaded from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection.ResultsAnalysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in Plasmodium vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5′ and 3′ regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5′ and 3′ regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population.ConclusionsHigh genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5′ and the 3′ regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi. No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi.

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.

Immunogenicity analysis of genetically conserved segments in Plasmodium ovale merozoite surface protein-8

BackgroundPlasmodium ovale is widely distributed across tropical regions and has two closely related but distinct species, namely P. ovale curtisi and P. ovale wallikeri. Combining genetic characterization with the immunogenicity of merozoite surface protein-8 (MSP-8) supports considering MSP-8 as a candidate target for blood-stage vaccines against malaria. However, no previous studies have focused on characterizing the genetic diversity and immunogenicity of PoMSP-8.MethodsBlood samples were collected from 42 patients infected with P. ovale. The patients were migrant workers returning to the Jiangsu Province from Africa; genomic DNA was extracted from their blood samples for sequencing and protein expression. The recombinant PoMSP-8 (rPoMSP-8) proteins were expressed and purified to assess their immune responses in BALB/c mice.ResultsThe sequences of the P. ovale curtisi and P. ovale wallikeri msp8 genes were completely conserved in each isolate. The rPoMSP-8 proteins were successfully expressed and purified as ~70 kDa proteins. Antibodies raised against rPoMSP-8 in mice showed appropriate immunoreactivity, as evidenced by immunoblotting. These specific antibodies were detected at day 7 post-immunization, and their levels increased throughout the whole immunization period. rPoMSP-8-raised antibodies had high endpoint titers (1:5,120,000) and high avidity (PocMSP-8: 94.84%, PowMSP-8: 92.69%). Cross-reactivity between rPocMSP-8 and rPowMSP-8 was observed with each anti-PoMSP8-specific antibody.ConclusionsRemarkable conservation and high immunogenicity was observed in both rPoMSP-8s. Intriguingly, cross-reaction between rPocMSP-8 and rPowMSP-8 was detected, suggesting that a single PoMSP8-based construction might be applicable for both species.

PO 8590 COMPARATIVE ANALYSIS OF IGG RESPONSES TO RECOMBINANT Qβ PHAGE DISPLAYED MSP3 AND UBO5 IN DUAL HIV/MALARIA-CO-INFECTED ADULTS

BackgroundImmunoglobulin G (IgG)-specific responses against Plasmodium falciparum merozoite antigens such as the merozoite surface protein 3 (MSP3) and UBO5 are known to play critical roles in parasitaemia control and protection from symptomatic illness. However, when there is intense perennial malaria transmission coupled with concurrent infection with the human immunodeficiency virus type 1 (HIV), knowledge of IgG antibody response profiles is limited.In this study we assessed the impact of dual HIV/malaria infections on IgG subclass responses to MSP3 (QβMSP3) and UBO5 (QβUB05) in individuals living in two areas of Cameroon differing in malaria transmission intensity.MethodsIgG and IgG subclass responses specific to either MSP3 or UBO5 were determined in plasma from study participant by ELISA. To improve reactivity with their respective antibodies the antigens were displayed upon the surface of the RNA coliphage Qβ.ResultsWe observed differences in antigen-specific IgG and IgG subclass responses which were dependent upon the antigen type, malaria transmission intensity, HIV infection, malaria infection and dual HIV/malaria infections. Individuals living in areas with high malaria transmission, had irrespective of HIV or malaria status significantly higher IgG responses to both antigens (p=0.0001 for QβMSP3, p=0.0001 for QβUB05) than their counterpart from areas with low transmission. When dual HIV/malaria infection is considered, significantly higher QβMSP3 specific IgG1 (p=0.0001) and IgG3 (p=0.04) responses in double-negative individuals was associated with protection against malaria in areas with low transmission. Superior QβUBO5 specific IgG1 responses (p=0.0001) in double-negative individuals were associated with protection in areas with high transmission in contrast to significantly higher IgG3 responses to QβUBO5 (p=0.0001) which were more relevant to protection in areas with low malaria transmission in the same population.ConclusionThus, understanding immune responses to QβUBO5 and QβMSP3 could facilitate the development of immunotherapeutic strategies suitable for areas differing in malaria transmission intensity.

Effect of low complexity regions within the PvMSP3\u03b1 block II on the tertiary structure of the protein and implications to immune escape mechanisms

BackgroundPlasmodium vivax merozoite surface protein 3α (PvMSP3α) is a promising vaccine candidate which has shown strong association with immunogenicity and protectiveness. Its use is however complicated by evolutionary plasticity features which enhance immune evasion. Low complexity regions (LCRs) provide plasticity in surface proteins of Plasmodium species, but its implication in vaccine design remain unexplored. Here population genetic, comparative phylogenetic and structural biology analysis was performed on the gene encoding PvMSP3α.ResultsThree LCRs were found in PvMSP3α block II. Both the predicted tertiary structure of the protein and the phylogenetic trees based on this region were influenced by the presence of the LCRs. The LCRs were mainly B cell epitopes within or adjacent. In addition a repeat motif mimicking one of the B cell epitopes was found within the PvMSP3a block II low complexity region. This particular B cell epitope also featured rampant alanine substitutions which might impair antibody binding.ConclusionThe findings indicate that PvMSP3α block II possesses LCRs which might confer a strong phenotypic plasticity. The phenomenon of phenotypic plasticity and implication of LCRs in malaria immunology in general and vaccine candidate genes in particular merits further exploration.

Transcriptome profiling of Plasmodium vivax in Saimiri monkeys identifies potential ligands for invasion

Unlike the case in Asia and Latin America, Plasmodium vivax infections are rare in sub-Saharan Africa due to the absence of the Duffy blood group antigen (Duffy antigen), the only known erythrocyte receptor for the P. vivax merozoite invasion ligand, Duffy binding protein 1 (DBP1). However, P. vivax infections have been documented in Duffy-negative individuals throughout Africa, suggesting that P. vivax may use ligands other than DBP1 to invade Duffy-negative erythrocytes through other receptors. To identify potential P. vivax ligands, we compared parasite gene expression in Saimiri and Aotus monkey erythrocytes infected with P. vivax Salvador I (Sal I). DBP1 binds Aotus but does not bind to Saimiri erythrocytes; thus, P. vivax Sal I must invade Saimiri erythrocytes independent of DBP1. Comparing RNA sequencing (RNAseq) data for late-stage infections in Saimiri and Aotus erythrocytes when invasion ligands are expressed, we identified genes that belong to tryptophan-rich antigen and merozoite surface protein 3 (MSP3) families that were more abundantly expressed in Saimiri infections compared with Aotus infections. These genes may encode potential ligands responsible for P. vivax infections of Duffy-negative Africans.

Inhibition of parasite invasion by monoclonal antibody against epidermal growth factor-like domain of Plasmodium vivax merozoite surface protein 1 paralog

The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P), which has epidermal growth factor (EGF)-like domains, was identified as a novel erythrocyte adhesive molecule. This EGF-like domain (PvMSP1P-19) elicited high level of acquired immune response in patients. Antibodies against PvMSP1P significantly reduced erythrocyte adhesion activity to its unknown receptor. To determine PvMSP1P-19-specific antibody function and B-cell epitopes in vivax patients, five monoclonal antibodies (mAbs) and 18-mer peptides were generated. The mAb functions were determined by erythrocyte-binding inhibition assay and invasion inhibition assay with P. knowlesi. B-cell epitopes of PvMSP1P-19 domains were evaluated by peptide microarray. The pvmsp1p-19 sequences showed limited polymorphism in P. vivax worldwide isolates. The 1BH9-A10 showed erythrocyte binding inhibitory by interaction with the N-terminus of PvMSP1P-19, while this mAb failed to recognize PkMSP1P-19 suggesting the species-specific for P. vivax. Other mAbs showed cross-reactivity with PkMSP1P-19. Among them, the 2AF4-A2 and 2AF4-A6 mAb significantly reduced parasite invasion through C-terminal recognition. The linear B-cell epitope in naturally exposed P. vivax patient was identified at three linear epitopes. In this study, PvMSP1P-19 N-terminal-specific 1BH9-A10 and C-terminal-specific 2AF4 mAbs showed functional activity for epitope recognition suggesting that PvMSP1P may be useful for vaccine development strategy for specific single epitope to prevent P. vivax invasion.

Genetic diversity and allelic variation in MSP3α gene of paired clinical Plasmodium vivax isolates from Delhi, India

BackgroundPlasmodium vivax malaria accounts for 80% of the malaria cases in Delhi, India. The gene merozoite surface protein 3 alpha (MSP3α) is highly polymorphic and has been used as marker in many P. vivax population studies. MethodsMSP3α has been used to assess the genetic diversity of P. vivax samples from Delhi (India) having more than one malaria episode (s) i.e. clinically identified relapse cases using PCR-RFLP and sequencing. ResultsThree major genotypes 2.0 kb (A), 1.4 kb (B) and 1.2 kb (C) were amplified from 72 isolates with frequencies of 72.2%, 19.44% and 9.72% respectively. One sample out of 72 showed mixed infection having both A and B type genotypes. 82.05% patients showed same genotype while only 17.94% patients showed different genotypes after subsequent malaria episodes. 18 different genotypes with Alu I and 35 with Hha I were identified among 72 samples analyzed by restriction fragment length polymorphism (RFLP). 18 Pvmsp3α nucleotide sequences were analyzed and it did not reveal any distinct intragenic differences within sequences of the same type, however, allelic diversity among the three types (Π = 0.029703) was observed. Phylogenetic analysis showed allelic family types A, B and C were not clustered but distributed in different branches. The results indicate that the P. vivax parasite population is highly diverse in Delhi, India. A large number of amino acid substitutions were found at the locus of the isolates when compared with the Belem Strain (Π = 0.030528). The substantial sequence diversity is largely restricted to certain domains of encoded protein. Analysis of synonymous and nonsynonymous substitutions suggested that different selection forces were operating on different regions of the protein molecule. ConclusionWe propose that genotyping of the PvMSP-3α gene as one of the molecular tools for differentiating relapse from new infection in epidemiological settings. The analyses of sequence polymorphism in PvMSP-3α gene enable it as potential candidate for inclusion in a P.vivax vaccine research.

Mosquito Bite-Induced Controlled Human Malaria Infection with Plasmodium vivax or P. falciparum Generates Immune Responses to Homologous and Heterologous Preerythrocytic and Erythrocytic Antigens.

Seroepidemiological studies on the prevalence of antibodies to malaria antigens are primarily conducted on individuals from regions of endemicity. It is therefore difficult to accurately correlate the antibody responses to the timing and number of prior malaria infections. This study was undertaken to assess the evolution of antibodies to the dominant surface antigens of Plasmodium vivax and P. falciparum following controlled human malaria infection (CHMI) in malaria-naive individuals. Serum samples from malaria-naive adults, collected before and after CHMI with either P. vivax (n = 18) or P. falciparum (n = 18), were tested for the presence of antibodies to the circumsporozoite protein (CSP) and the 42-kDa fragment of merozoite surface protein 1 (MSP-142) of P. vivax and P. falciparum using an enzyme-linked immunosorbent assay (ELISA). Approximately 1 month following CHMI with either P. vivax or P. falciparum, >60% of subjects seroconverted to homologous CSP and MSP-1. More than 50% of the subjects demonstrated reactivity to heterologous CSP and MSP-142, and a similar proportion of subjects remained seropositive to homologous MSP-142 >5 months after CHMI. Computational analysis provides insight into the presence of cross-reactive responses. The presence of long-lived and heterologous reactivity and its functional significance, if any, need to be taken into account while evaluating malaria exposure in field settings.

Comparative Analysis of Igg Responses to Recombinant Qβ Phage Displayed MSP3 and UB05 in Dual HIV-Malaria Infected Adults Living in areas differing in Malaria Transmission Intensities

Immunoglobulin G (IgG) specific responses against Plasmodium falciparum merozoite antigens such as the merozoite surface protein 3 (MSP3) and UB05 are known to play critical roles in parasiteamia control and protection from symptomatic illness. However when there is intense perennial malaria transmission coupled with concurrent infection with the human immunodeficiency virus type 1 (HIV), knowledge of IgG antibody response profiles is limited. In this study we assessed the impact of dual HIV-Malaria infections on IgG subclass responses to MSP3 (QβMSP3) and UB05 (QβUB05) in individuals living in two areas of Cameroon differing in malaria transmission intensity. We observed differences in antigen specific IgG and IgG subclass responses which were dependent upon the antigen type, malaria transmission intensity, HIV infection, malaria infection and dual HIV-malaria infections. Individuals living in high malaria transmission areas irrespective of HIV or malaria status had significantly higher IgG responses to both antigens (P=0.0001 for QβMSP3, P=0.0001 for QβUB05) than their counterpart from low transmission areas. When dual HIV-Malaria infection is considered significantly higher QβMSP3 specific IgG1 (P=0.0001) and IgG3 (P=0.04) responses in double negative individuals was associated with protection against malaria in low transmission areas. Increased QβUB05 specific IgG1 responses (P=0.0001) in double negative individuals were associated with protection in high transmission areas in contrast to significantly higher IgG3 responses to QβUB05 (P=0.0001) which were more relevant to protection in low malaria transmission areas in the same population. These findings imply that QβMSP3 might not be suitable as a standalone vaccine in areas differing in transmission intensity. However, antigenicity of UB05 most likely predicts immunity in both low and high transmission areas and could be used either alone or in combination with other antigens for vaccine studies in areas differing in transmission intensities. Understanding immune responses to QβUB05 and QβMSP3 could thus enable the development of efficacious vaccines or commensurate immunotherapeutic strategies suitable for areas differing in malaria transmission intensity.

The impact of early life exposure to Plasmodium falciparum on the development of naturally acquired immunity to malaria in young Malawian children

BackgroundAntibodies targeting malaria blood-stage antigens are important targets of naturally acquired immunity, and may act as valuable biomarkers of malaria exposure.MethodsSix-hundred and one young Malawian children from a randomized trial of prenatal nutrient supplementation with iron and folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient supplements were followed up weekly at home and febrile episodes were investigated for malaria from birth to 18 months of age. Antibodies were measured for 601 children against merozoite surface proteins (MSP1 19kD, MSP2), erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2 (Rh2A9), schizont extract and variant surface antigens expressed by Plasmodium falciparum-infected erythrocytes (IE) at 18 months of age. The antibody measurement data was related to concurrent malaria infection and to documented episodes of clinical malaria.ResultsAt 18 months of age, antibodies were significantly higher among parasitaemic than aparasitaemic children. Antibody levels against MSP1 19kD, MSP2, schizont extract, and IE variant surface antigens were significantly higher in children who had documented episodes of malaria than in children who did not. Antibody levels did not differ between children with single or multiple malaria episodes before 18 months, nor between children who had malaria before 6 months of age or between 6 and 18 months.ConclusionsAntibodies to merozoite and IE surface antigens increased following infection in early childhood, but neither age at first infection nor number of malaria episodes substantially affected antibody acquisition. These findings have implications for malaria surveillance during early childhood in the context of elimination.Trials registration Clinical Trials Registration: NCT01239693 (Date of registration: 11-10-2010). URL: http://www.ilins.org