Plasmodium Vivax Circumsporozoite Protein Research Articles

Poly I:C elicits broader and stronger humoral and cellular responses to a Plasmodium vivax circumsporozoite protein malaria vaccine than Alhydrogel in mice.

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents Plasmodium falciparum (Pf) malaria but is ineffective against Plasmodium vivax (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria.

Production and Purification of Plasmodium Circumsporozoite Protein in Lactococcus lactis.

Malaria is a vector-borne disease caused by Plasmodium parasites of which Plasmodium falciparum contributed to an estimated 247 million cases worldwide in 2021 (WHO malaria report 2022). The P. falciparum Circumsporozoite protein (PfCSP) covers the surface of the sporozoite which is critical to cell invasion in the human host. PfCSP is the leading pre-erythrocytic vaccine candidate and forms the basis of the RTS'S (Mosquirix®) malaria vaccine. However, high-yield production of full-length PfCSP with proper folding has been challenging. Here, we describe expression and purification of full-length PfCSP (containing 4 NVDP and 38 NANP repeats) with proper conformation by a simple three-step procedure in the Lactococcus lactis expression system.

The αTSR Domain of Plasmodium Circumsporozoite Protein Bound Heparan Sulfates and Elicited High Titers of Sporozoite Binding Antibody After Displayed by Nanoparticles.

Malaria is a devastating infectious illness caused by protozoan Plasmodium parasites. The circumsporozoite protein (CSP) on Plasmodium sporozoites binds heparan sulfate proteoglycan (HSPG) receptors for liver invasion, a critical step for prophylactic and therapeutic interventions. In this study, we characterized the αTSR domain that covers region III and the thrombospondin type-I repeat (TSR) of the CSP using various biochemical, glycobiological, bioengineering, and immunological approaches. We found for the first time that the αTSR bound heparan sulfate (HS) glycans through support by a fused protein, indicating that the αTSR is a key functional domain and thus a vaccine target. When the αTSR was fused to the S domain of norovirus VP1, the fusion protein self-assembled into uniform S60-αTSR nanoparticles. Three-dimensional structure reconstruction revealed that each nanoparticle consists of an S60 nanoparticle core and 60 surface displayed αTSR antigens. The nanoparticle displayed αTSRs retained the binding function to HS glycans, indicating that they maintained authentic conformations. Both tagged and tag-free S60-αTSR nanoparticles were produced via the Escherichia coli system at high yield by scalable approaches. They are highly immunogenic in mice, eliciting high titers of αTSR-specific antibody that bound specifically to the CSPs of Plasmodium falciparum sporozoites at high titer. Our data demonstrated that the αTSR is an important functional domain of the CSP. The S60-αTSR nanoparticle displaying multiple αTSR antigens is a promising vaccine candidate potentially against attachment and infection of Plasmodium parasites.

Molecular identification of vivax malaria relapse patients in the Yunnan Province based on homology analysis of the Plasmodium vivax circumsporozoite protein gene

More than 85% of the malaria burden in the Yunnan Province is caused by imported vivax malaria, and Yunnan is also where the majority of vivax malaria patients are diagnosed in China. Timely removal of the infection sources of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To that end, blood samples were collected from cases diagnosed and revalidated as single species infection with P. vivax in the Yunnan Province from 2013 to 2020. Specifically, samples from vivax malaria patients with suspected relapses episodes were subjected to PCR amplification, product sequencing, and analysis of the P. vivax circumsporozoite protein (pvcsp) gene. In total, 77 suspected relapse patients were identified out of 2484 cases infected with P. vivax, with a total of 81 recurrent episodes. A total of 156 CDS (coding DNA sequence) chains were obtained through PCR amplification and sequencing of the pvcsp gene from 159 blood samples, 121 of which can be matched to the paired sequences of 59 vivax malaria patients with both primary attack and recurrent experience. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites (VS), meaning every two paired sequence was completely homologous. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, indicating that the paired sequences was “weakly heterologous” with no fragment insertions (or deletions). All 59 vivax malaria patients with recurrences were caused by the activation of P. vivax hypnozoites originated from the same population as the primary infection. The paired analysis of the similarity between high variant genes allowed the identification of relapse episodes caused by P. vivax homologous hypnozoites and also demonstrated pvcsp gene as one of the candidate molecular markers for tracing infection origin.

Persistently high proportions of plasmodium-infected Anopheles funestus mosquitoes in two villages in the Kilombero valley, South-Eastern Tanzania

BackgroundIn south-eastern Tanzania where insecticide-treated nets have been widely used for >20 years, malaria transmission has greatly reduced but remains highly heterogenous over small distances. This study investigated the seasonal prevalence of Plasmodium sporozoite infections in the two main malaria vector species, Anopheles funestus and Anopheles arabiensis for 34 months, starting January 2018 to November 2020. MethodsAdult mosquitoes were collected using CDC-light traps and Prokopack aspirators inside local houses in Igumbiro and Sululu villages, where earlier surveys had found very high densities of An. funestus. Collected females were sorted by taxa, and the samples examined using ELISA assays for detecting Plasmodium circumsporozoite protein in their salivary glands. ResultsOf 7859 An. funestus tested, 4.6% (n = 365) were positive for Pf sporozoites in the salivary glands. On the contrary, only 0.4% (n = 9) of the 2382 An. arabiensis tested were positive. The sporozoite prevalence did not vary significantly between the villages or seasons. Similarly, the proportions of parous females of either species were not significantly different between the two villages (p > 0.05) but was slightly higher in An. funestus (0.50) than in An. arabiensis (0.42). Analysis of the 2020 data determined that An. funestus contributed 97.7% of all malaria transmitted in households in these two villages. ConclusionsIn contexts where individual vector species mediate most of the pathogen transmission, it may be most appropriate to pursue a species-focused approach to better understand the ecology of the dominant vectors and target them with effective interventions to suppress transmission. Despite the ongoing efforts on tackling malaria in the two study villages, there is still persistently high Plasmodium infection prevalence in local populations of An. funestus, which now carry ~97% of all malaria infections and mediates intense year-round transmission. Further reduction in malaria burden in these or other similar settings requires effective targeting of An. funestus.

Immune System Modulation by the Adjuvants Poly (I:C) and Montanide ISA 720.

Adjuvants are essential for vaccine development, especially subunit-based vaccines such as those containing recombinant proteins. Increasing the knowledge of the immune response mechanisms generated by adjuvants should facilitate the formulation of vaccines in the future. The present work describes the immune phenotypes induced by Poly (I:C) and Montanide ISA 720 in the context of mice immunization with a recombinant protein based on the Plasmodium vivax circumsporozoite protein (PvCSP) sequence. Mice immunized with the recombinant protein plus Montanide ISA 720 showed an overall more robust humoral response, inducing antibodies with greater avidity to the antigen. A general trend for mixed Th1/Th2 inflammatory cytokine profile was increased in Montanide-adjuvanted mice, while a balanced profile was observed in Poly (I:C)-adjuvanted mice. Montanide ISA 720 induced a gene signature in B lymphocytes characteristic of heme biosynthesis, suggesting increased differentiation to Plasma Cells. On the other hand, Poly (I:C) provoked more perturbations in T cell transcriptome. These results extend the understanding of the modulation of specific immune responses induced by different classes of adjuvants, and could support the optimization of subunit-based vaccines.

Hepatotropic Peptides Grafted onto Maleimide-Decorated Nanoparticles: Preparation, Characterization and In Vitro Uptake by Human HepaRG Hepatoma Cells.

We recently demonstrated the strong tropism of George Baker (GB) Virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB) derived synthetic peptides towards hepatoma cells. In a first approach, these peptides were covalently bound to poly(benzyl malate) (PMLABe73) and poly(ethylene glycol)-block-PMLABe73 (PEG62-b-PMLABe73) (co)polymers, and corresponding peptide-decorated nanoparticles (NPs) were prepared by nanoprecipitation. We showed that peptide enhanced NPs internalization by hepatoma cells. In the present work, we set up a second strategy to functionalize NPs prepared from PMLABe73 derivates. First, maleimide-functionalized PMLABe73 (Mal-PMLABe73) and PEG62-b-PMLABe73 (Mal-PEG62-b-PMLABe73) were synthesized and corresponding NPs were prepared by nanoprecipitation. Then, peptides (GBVA10-9, CPB and their scramble controls GBVA10-9scr and CPBscr) with a thiol group were engrafted onto the NPs’ maleimide groups using the Michael addition to obtain peptide functionalized NPs by post-formulation procedure. These peptide-modified NPs varied in diameter and dispersity depending on the considered peptides and/or (co)polymers but kept their spherical shape. The peptide-functionalized NPs were more efficiently internalized by HepaRG hepatoma cells than native and maleimide-NPs with various levels relying on the peptide’s nature and the presence of PEG. We also observed important differences in internalization of NPs functionalized by the maleimide-thiol-peptide reaction compared to that of NPs prepared from peptide-functionalized PMLABe73 derivatives.

Randomized clinical trial to assess the protective efficacy of a Plasmodium vivax CS synthetic vaccine

A randomized, double-blind, controlled vaccine clinical trial was conducted to assess, as the primary outcome, the safety and protective efficacy of the Plasmodium vivax circumsporozoite (CS) protein in healthy malaria-naïve (phase IIa) and semi-immune (phase IIb) volunteers. Participants (n = 35) were randomly selected from a larger group (n = 121) and further divided into naïve (n = 17) and semi-immune (n = 18) groups and were immunized at months 0, 2, and 6 with PvCS formulated in Montanide ISA-51 adjuvant or placebo (adjuvant alone). Specific antibodies and IFN-γ responses to PvCS were determined as secondary outcome; all experimental volunteers developed specific IgG and IFN-γ. Three months after the last immunization, all participants were subjected to controlled human malaria infection. All naive controls became infected and drastic parasitemia reduction, including sterile protection, developed in several experimental volunteers in phase IIa (6/11) (54%, 95% CI 0.25–0.84) and phase IIb (7/11) (64%, 95% CI 0.35–0.92). However, no difference in parasitemia was observed between the phase IIb experimental and control subgroups. In conclusion, this study demonstrates significant protection in both naïve and semi-immune volunteers, encouraging further PvCS vaccine clinical development. Trial registration number NCT 02083068. This trial was funded by Colciencias (grant 529-2009), NHLBI (grant RHL086488 A), and MVDC/CIV Foundation (grant 2014-1206).

Plasmodium Circumsporozoite Protein Enhances the Efficacy of Gefitinib in Lung Adenocarcinoma Cells by Inhibiting Autophagy via Proteasomal Degradation of LC3B.

Background: Almost all lung adenocarcinoma (LUAD) patients with EGFR mutant will develop resistance to EGFR-TKIs, which limit the long-term clinical application of these agents. Accumulating evidence shows one of the main reasons for resistance to EGFR-TKIs is induction of autophagy in tumor cells. Our previous study found that circumsporozoite protein (CSP) in Plasmodium can suppress autophagy in host hepatocytes. However, it is unknown whether CSP-mediated inhibition of autophagy could improve the anti-tumor effect of EGFR-TKIs. Methods: We constructed A549 and H1975 cell lines with stable overexpression of CSP (OE-CSP cells). CCK-8, Lactate Dehydrogenase (LDH), flow cytometry, and colony analysis were performed to observe the effect of CSP overexpression on cell viability, apoptosis rate, and colony formation ratio. The sensitizing effect of CSP on gefitinib was evaluated in vivo using a subcutaneous tumor model in nude mice and immunohistochemical assay. The role of CSP in regulation of autophagy was investigated by laser confocal microscopy assay and western blotting. A transcriptome sequencing assay and real-time polymerase chain reaction were used to determine the levels of mRNA for autophagy-related proteins. Cycloheximide (CHX), MG132, TAK-243, and immunoprecipitation assays were used to detect and confirm proteasomal degradation of LC3B. Results: OE-CSP A549 and H1975 cells were more sensitive to gefitinib, demonstrating significant amounts of apoptosis and decreased viability. In the OE-CSP group, autophagy was significantly inhibited, and there was a decrease in LC3B protein after exposure to gefitinib. Cell viability and colony formed ability were recovered when OE-CSP cells were exposed to rapamycin. In nude mice with xenografts of LUAD cells, inhibition of autophagy by CSP resulted in suppression of cell growth, and more marked apoptosis during exposure to gefitinib. CSP promoted ubiquitin-proteasome degradation of LC3B, leading to inhibition of autophagy in LUAD cells after treatment with gefitinib. When LUAD cells were treated with ubiquitin activating enzyme inhibitor TAK-243, cell viability, apoptosis, and growth were comparable between the OE-CSP group and a control group both in vivo and in vitro. Conclusion: CSP can inhibit gefitinib-induced autophagy via proteasomal degradation of LC3B, which suggests that CSP could be used as an autophagy inhibitor to sensitize EGFR-TKIs.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay.

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Adaptation of ELISA detection of Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in mosquitoes to a multiplex bead-based immunoassay

BackgroundPlasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites.MethodsRecombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins.ResultsFollowing optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined.ConclusionsThe csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Seroprevalence of Plasmodium vivax Circumsporozoite Protein Antibody in High-Risk Malaria Areas in Korea.

The circumsporozoite protein (CSP) of Plasmodium spp. is a diagnostic antigen and useful biomarker for monitoring short-term/seasonal changes to malaria transmission. Using P. vivax CSP antibody ELISA, epidemiological characteristics were analyzed in the residents of Ganghwa, Cheorwon, Paju, and Goseong from 2017 to 2018. In Ganghwa and Cheorwon, 1.6% and 1.2% of residents, respectively, were PvCSP-antibody-positive in 2018, which indicates a decrease of 0.4% in the positive rate compared to 2017. The annual parasite incidence (API) in Ganghwa and Cheorwon was 24.9 and 10.5 in 2017 and 20.3 and 10.7 in 2018, respectively. Although the changes were not significant, the API in Ganghwa decreased slightly by 4.5 in 2018 compared to the previous year. In Paju and Goseong, 3.9% and 2.0% of residents were positive for the PvCSP antibody. The API in Paju was 13.1 in 2017 and 16.0 in 2018, although no malaria patients were reported for the 2 years. Therefore, the results suggest that PvCSP is a useful antigen for confirming initial malaria infection. Additionally, considering that the antibody is relatively transient, it can be employed for sero-epidemiological studies to determine the extent of malaria transmission in the current year.

Synthesis of Poly(Malic Acid) Derivatives End-Functionalized with Peptides and Preparation of Biocompatible Nanoparticles to Target Hepatoma Cells.

Recently, short synthetic peptides have gained interest as targeting agents in the design of site-specific nanomedicines. In this context, our work aimed at developing new tools for the diagnosis and/or therapy of hepatocellular carcinoma (HCC) by grafting the hepatotropic George Baker (GB) virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB)-derived peptides to the biocompatible poly(benzyl malate), PMLABe. We successfully synthesized PMLABe derivatives end-functionalized with peptides GBVA10-9, CPB, and their corresponding scrambled peptides through a thiol/maleimide reaction. The corresponding nanoparticles (NPs), varying by the nature of the peptide (GBVA10-9, CPB, and their scrambled peptides) and the absence or presence of poly(ethylene glycol) were also successfully formulated using nanoprecipitation technique. NPs were further characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS) and transmission electron microscopy (TEM), highlighting a diameter lower than 150 nm, a negative surface charge, and a more or less spherical shape. Moreover, a fluorescent probe (DiD Oil) has been encapsulated during the nanoprecipitation process. Finally, preliminary in vitro internalisation assays using HepaRG hepatoma cells demonstrated that CPB peptide-functionalized PMLABe NPs were efficiently internalized by endocytosis, and that such nanoobjects may be promising drug delivery systems for the theranostics of HCC.

Avid binding by B cells to the Plasmodium circumsporozoite protein repeat suppresses responses to protective subdominant epitopes.

SummaryAntibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSPRepeat) can protect against malaria. However, it has also been suggested that the CSPRepeat is a decoy that prevents the immune system from mounting responses against other domains of CSP. Here, we show that, following parasite immunization, B cell responses to the CSPRepeat are immunodominant over responses to other CSP domains despite the presence of similar numbers of naive B cells able to bind these regions. We find that this immunodominance is driven by avid binding of the CSPRepeat to cognate B cells that are able to expand at the expense of B cells with other specificities. We further show that mice immunized with repeat-truncated CSP molecules develop responses to subdominant epitopes and are protected against malaria. These data demonstrate that the CSPRepeat functions as a decoy, but truncated CSP molecules may be an approach for malaria vaccination.

Evaluation of Combinatory Effects of Plasmodium Circumsporozoite Protein and Complement Regulatory Protein Expression of Recombinant Baculovirus Vectors.

Baculovirus vectors (BVs) are safely able to transduce foreign genes and express them in mammalian cells. However, the transduction activity of BVs is strongly reduced by the attack of serum complement, which is one of the major obstacles in the use of BVs for in vivo gene transfer. One strategy to overcome this problem is the display of complement regulatory proteins (CRPs) on BV virions. We previously developed CD46-decay accelerating factor (DAF)-CD59 triple fusion type BV showing potent complement resistance. We also developed BVs expressing Plasmodium circumsporozoite protein (CSP) to enhance transduction efficacy in hepatic cells. In this study, we investigated the combination of CSP and CRPs in a BV system to evaluate transduction efficacy along with complement resistance. To accomplish the combination of CSP and CRPs, we generated insect Sf9 cells stably expressing CRPs, to which CSP type BV was infected. The BVs collected from these infected cells were confirmed to possess both CSP and CRPs in virions. We demonstrated that CSP-CD46-DAF-CD59 type BV, containing both CSP and CD46-DAF-CD59, showed a significant increase in transduction efficacy in human hepatoma HepG2 cells under intact serum exposure compared with control type BV or CSP type BV, retaining both advantages of CSP and CD46-DAF-CD59. Collectively, these results demonstrated that the utilization of stably expressing Sf9 cells to introduce the protein products of interest, e.g., CRPs into BVs, would be useful strategy to generate BVs with novel functions such as resistance against serum complement attack.

Genotyping and Phylogenetic Analysis of Plasmodium vivax Circumsporozoite Protein (pvcsp) Gene of Clinical Isolates in South-Eastern Iran

Circumsporozoite protein (CSP) is one of the most important surface sporozoite antigens in malaria, recently considered as a candidate for vaccination. Considering the importance of CSP, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax Circumsporozoite Protein (Pvcsp) in the southeastern region of Iran during 2015-2016. To investigate polymorphism and genetic diversity, 20 blood samples were collected from patients with P. vivax, then DNA was extracted and amplified using partial sequence of CSP gene. Polymerase chain reaction (PCR) products were sequenced and compared to sequences from genomic databases using BLAST. Genetic evaluation and phylogenic analysis were performed using MEGA7 and DnaSP5 software's on 38 sequences include 20 sequences of our study and 18 sequences of Gene Bank. Eleven isolates were VK210 genotype and 9 isolates contained VK247. The result of variable segregation nucleotide site indicated that the differentiation of sequences in CSP were 25.67% in our 20 samples which are less than the 38 samples with a value of 26.67%. Comparing the ratio of dN/dS regions in the CSP gene indicates that the CSP varies more synonymously and amino acid has lower variation. Out of 38 samples, 35 unique haplotypes were identified based on 1042 nucleotide sequences in CSP, showing a variation percentage of 99.4%. The Tajima D analyses showed that CSP gene in P. vivax had a positive number in the total analyzed sequences, which means that the P. vivax mutations are in order to select positive evolution.

Protective efficacy of peptides from Plasmodium vivax circumsporozoite protein

Vivax malaria is a major cause of morbidity and mortality worldwide, with several million clinical cases per year and 2.5 billion at risk of infection. A vaccine is urgently needed but the most advanced malaria vaccine, VMP001, confers only very low levels of protection against vivax malaria challenge in humans. VMP001 is based on the circumsporozoite protein (CSP) of Plasmodium vivax. Here a virus-like particle, Qβ, is used as a platform to generate very high levels of antibody against peptides from PvCSP in mice, in order to answer questions important to further development of P. vivax CSP (PvCSP) vaccines. Minimal peptides from the VK210 and VK247 allelic variants of PvCSP are found to be highly protective as Qβ-peptide vaccines, using transgenic P. berghei parasites expressing the homologous PvCSP allelic variant. A target of neutralising antibodies within the nonamer unit repeat of VK210, AGDR, is found, as a Qβ-peptide vaccine, to provide partial protection against malaria challenge, and enhances protective efficacy when combined with full-length PvCSP vaccination. A truncated form of PvCSP, missing the N-terminal domain, is found to confer much higher levels of protective efficacy than full-length PvCSP. Peptides derived from highly conserved areas of PvCSP, RI and RII, are found not to confer protective efficacy as Qβ-peptide vaccines.

Analysis of anti-Plasmodium IgG profiles among Fulani nomadic pastoralists in northern Senegal to assess malaria exposure

BackgroundNorthern Senegal is a zone of very low malaria transmission, with an annual incidence of < 5/1000 inhabitants. This area, where the Senegal National Malaria Control Programme has initiated elimination activities, hosts Fulani, nomadic, pastoralists that spend the dry season in the south where malaria incidence is higher (150–450/1000 inhabitants) and return to the north with the first rains. Previous research demonstrated parasite prevalence of < 1% in this Fulani population upon return from the south, similar to that documented in the north in cross-sectional surveys.MethodsA modified snowball sampling survey of nomadic pastoralists was conducted in five districts in northern Senegal during September and October 2014. Demographic information and dried blood spots were collected. Multiplex bead-based assays were used to assess antibody responses to merozoite surface protein (MSP-119) antigen of the four primary Plasmodium species, as well as circumsporozoite protein (CSP) and liver stage antigen (LSA-1) of Plasmodium falciparum.ResultsIn the five study districts, 1472 individuals were enrolled, with a median age of 22 years (range 1 to 80 years). Thirty-two percent of subjects were under 14 years and 57% were male. The overall seroprevalence of P. falciparum MSP-119, CSP and LSA-1 antibodies were 45, 12 and 5%, respectively. Plasmodium falciparum MSP-119 antibody responses increased significantly with age in all study areas, and were significantly higher among males. The highest seroprevalence to P. falciparum antigens was observed in the Kanel district (63%) and the lowest observed in Podor (28%). Low seroprevalence was observed for non-falciparum species in all the study sites: 0.4, 0.7 and 1.8%, respectively, for Plasmodium ovale, Plasmodium vivax and Plasmodium malariae MSP-1. Antibody responses to P. vivax were observed in all study sites except Kanel.ConclusionPrevalence of P. falciparum MSP-119 antibodies and increases by study participant age provided data for low levels of exposure among this transient nomadic population. In addition, antibody responses to P. falciparum short half-life markers (CSP and LSA-1) and non-falciparum species were low. Further investigations are needed to understand the exposure of the Fulani population to P. vivax.

Optimization of an in vivo model to study immunity to Plasmodium falciparum pre-erythrocytic stages

BackgroundThe circumsporozoite protein (CSP) of Plasmodium is a key surface antigen that induces antibodies and T-cells, conferring immune protection in animal models and humans. However, much of the work on CSP and immunity has been developed based on studies using rodent or non-human primate CSP antigens, which may not be entirely translatable to CSP expressed by human malaria parasites, especially considering the host specificity of the different species.MethodsUsing a genetically engineered strain of Plasmodium berghei that expresses luciferase, GFP and the Plasmodium falciparum orthologue of CSP, the effect of laboratory preparation, mosquito treatment and mouse factors on sporozoite infectivity was assessed using an in vivo bioluminescence assay on mice. This assay was compared with a PCR-based protection assay using an already described monoclonal antibody that can provide sterile protection against sporozoite challenge.ResultsBioluminescence assay demonstrated similar detection levels of the quantity and kinetics of liver-stage infection, compared to PCR-based detection. This assay was used to evaluate treatment of sporozoite and delivery method on mouse infectivity, as well as the effects of age, sex and strain of mice. Finally, this assay was used to test the protective capacity of monoclonal antibody AB317; results strongly recapitulate the findings of previous work on this antibody.ConclusionsThe PbGFP-Luc line and in vivo bioluminescence imaging provide highly sensitive read-outs of liver-stage infection in mice, and this method can be useful to reliably evaluate potency of pre-erythrocytic interventions.

Exploration of genetic diversity of Plasmodium vivax circumsporozoite protein (Pvcsp) and Plasmodium vivax sexual stage antigen (Pvs25) among North Indian isolates

BackgroundMalaria is one of the important vector-borne diseases with high fatality rates in tropical countries. The pattern of emergence and spread of novel antigenic variants, leading to escape of vaccine-induced immunity might be factors responsible for severe malaria. A high level of polymorphism has been reported among malarial antigens which are under selection pressure imposed by host immunity. There are limited reports available on comparative stage-specific genetic diversity among Plasmodium vivax candidate genes in complicated vivax malaria. The present study was planned to study genetic diversity (Pvcsp and Pvs25) among complicated and uncomplicated P. vivax isolates.MethodsPvcsp and Pvs2-specific PCRs and DNA sequencing were performed on P. vivax PCR positive samples. Genetic diversity was analysed using appropriate software.ResultsThe present study was carried out on 143 P. vivax clinical isolates, collected from Postgraduate Institute of Medical Education and Research, Chandigarh. Among the classic and variant types of Pvcsp, the VK210 (99%; 115/116) was found to be predominant in both complicated and uncomplicated group isolates. Out of the various peptide repeat motifs (PRMs) observed, GDRADGQPA (PRM1) and GDRAAGQPA (PRM2) was the most widely distributed among the P. vivax isolates. Whereas among the Pvs25 isolates, 100% of double mutants (E97Q/I130T) in both the complicated (45/45) as well as in the uncomplicated (81/81) group was observed.ConclusionAn analysis of genetic variability enables an understanding of the role of genetic variants in severe vivax malaria.