Plasmodium Vivax Circumsporozoite Research Articles

Protein-energetic malnutrition hinders malaria vaccine-derived cellular and class-switched antibody responses against the Plasmodium vivax circumsporozoite protein in mice.

Malaria continues to afflict hundreds of millions of lives annually, causing substantial fatalities despite available vaccines endorsed by the World Health Organization (WHO). However, these vaccines lack efficacy against Plasmodium vivax (Pv). Concomitantly, a considerable part of residents from several Pv-endemic areas face malnutrition, compromising their immunity to diseases, including malaria. Since our group developed an immunogenic yeast-expressing recombinant Pv circumsporozoite protein (yPvCSP-AllCT epitopes) capable of protecting mice against lethal transgenic parasites, we investigated the influence of malnutrition on vaccine-derived responses in C57Bl/6 mice. Animals subjected to a protein-restricted diet presented protein-energetic malnutrition, diminished vaccine-specific IgG-secreting cells in the bone marrow, and reduced IgG and IgG1 serum titers compared to mice under a control diet. IgM titers remained consistent across groups, suggesting that the nutrition status may influence the antibody affinity maturation. These findings emphasize the pivotal role of proper nutrition in enhancing vaccination immunity against Pv malaria.

A survey of malaria vectors feeding preference, biting site and resting behaviour in the malaria elimination settings of Dembiya District, north-western Ethiopia.

Despite the progress in scaling vector control interventions in Ethiopia, malaria is still a major health problem in the country. Monitoring of the local vector populations and the effectiveness of vector control strategies is necessary to guide programme decisions to optimize malaria prevention efforts. This study investigated the feeding preference, the biting behaviour and resting behaviours of Anopheles mosquitoes in selected localities of Dembiya District. Adult Anopheles mosquitoes were sampled indoors and outdoors from June 2018 to May 2019 using CDC light traps, pyrethrum spray catches, artificial pit shelters, and mouth aspirators at both Guramba Bata and Arebiya study sites. Anopheles mosquitoes were identified to the species level. Their blood meal source and Plasmodium sporozoite infections were determined using an enzyme-linked immunosorbent assay. Anopheles mosquitoes belonging to 11 species were identified from 2,055 collected mosquito specimens. Anopheles pharoensis was the predominant species at both the Guramba Bata (46.5%) and Arebiya (46.2%) study sites. The CDC light traps caught the highest number of Anopheles mosquitoes in both study sites. In Guramba Bata the density of outdoor host-seeking and resting Anopheles mosquitoes were higher than indoors (P ≤ 0.05). The human blood indexes (HBI) of indoor and outdoor host-seeking Anopheles arabiensis were 17.4% and 15.3%, respectively. The entomological inoculation rate (EIR) of outdoor host-seeking An. arabiensis was 4.7 infective bites/person/year. Additionally, the outdoor EIR of host-seeking Anopheles coustani was 25.7ib/p/year. Anopheles mosquitoes in Dembiya district were more likely to seek a host and rest outdoors than indoors. A reevaluation of vector control strategies is needed to ensure Ethiopia remains on the path to malaria elimination. The detection of Plasmodium circumsporozoite protein in potential secondary vectors, such as An. coustani requires further investigation to substantiate their role in malaria transmission.

Low Genetic Diversity of Plasmodium vivax Circumsporozoite Surface Protein in Clinical Isolates from Southern Thailand.

The genetic diversity within the circumsporozoite surface protein (PvCSP) of Plasmodium vivax, the predominant malaria species in Thailand, is primarily observed in the northwestern region along the Thailand-Myanmar border. However, as P. vivax cases shift to southern provinces, particularly Yala Province near the Thailand-Malaysia border, PvCSP diversity remains understudied. Between 2018 and 2020, 89 P. vivax isolates were collected in Yala Province, a significant malaria hotspot. Employing polymerase chain reaction amplification, restriction fragment length polymorphism (PCR-RFLP), and DNA sequencing, the gene encoding PvCSP (Pvcsp) was analyzed. All Yala P. vivax isolates belonged to the VK210 type, distinct from strains in the western region near the Myanmar border. The central repeat region of Pvcsp revealed two common peptide repeat motifs-GDRADGQPA and GDRAAGQPA-across all southern isolates. Sequence analysis identified two subtypes, with S1 more prevalent (92%) than S2 (8%). This study underscores the limited diversity of VK210 variants of P. vivax populations in southern Thailand. These baseline findings facilitate monitoring for potential new parasite variants, aiding in the future control and management of P. vivax in the region.

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.

Identification of immunodominant epitopes in allelic variants VK210 and VK247 of Plasmodium Vivax Circumsporozoite immunogen.

Plasmodium vivax-induced malaria is among the leading causes of morbidity and mortality in sub-tropical and tropical regions and infect 2.85 billion people globally. The continual rise and propagation of resistance against anti-malarial drugs is a prerequisite to develop a potent vaccine candidate for Plasmodium vivax (P. vivax). Circumsporozoite protein (CSP) is an important immunogen of malaria parasite that has the conserved CSP structure as an immune dominant B-cell epitope. In current study, we focused on designing multi-epitope vaccines (MEVs) using various immunoinformatics tools against Pakistani based allelic variants VK210 and VK247 of P. vivax CSP (PvCSP) gene. Antigenicity, allergic potential and physicochemical parameters of both PvCSP variants were assessed for the designed MEVs and they were within acceptable range suitable for post experimental investigations. The three-dimensional structures of both MEVs have been predicted ab initio, optimized, and validated by using different online servers. The both MEVs candidates were stable and free from aggregation-prone regions. The stability of both MEVs had been improved by a disulfide engineering approach. To estimate the binding energy and stability of the MEVs, molecular docking simulation and binding free energy calculations with TLR-4 immune receptor have been conducted. The docking score of PvCSP210 and PvCSP247 for TLR-4 was -6.34kJ/mol and-2.3kJ/mol, respectively. For PvCSP210-TLR4 system, mean RMSD was 4.96Å while PvCSP247-TLR4 system, average RMSD was 4.49Å. The binding free energy of PvCSP210-TLR4 complex and PvCSP247-TLR4 complex was -50.49/-117.15kcal/mol (MMGBSA/MMPSA) and -52.94/-96.26kcal/mol (MMGBSA/MMPSA), respectively. The expression of both MEVs produced in Escherichia coli K12 expression system by in silico cloning was significant. Immune simulation revealed that the proposed MEVs induce strong humoral and cellular immunological responses, in addition to significant production of interleukins and cytokines. In conclusions, we believed that the MEVs proposed in current research, using combine approach of immunoinformatics, structural biology and biophysical approaches, could induce protective and effective immune responses against P. vivax and the experimental validation of our findings could contribute to the development of potential malaria vaccine.

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.

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.

Prevalence of sibling-species of Anopheles (Cellia) fluviatilis complex in Himachal Pradesh, India.

Malaria is one of the most infectious and life-threatening vector borne disease in the tropics. Climate change can significantly influence malaria epidemiology and expansion of malaria vectors to hilly regions of Himachal Pradesh in India, hitherto considered areas of low transmission. Entomological surveillance in Kangra district of Himachal Pradesh revealed high density of a proven efficient vector of malaria, Anopheles fluviatilis, but transmission intensity of malaria was found very low. It was therefore considered prudent to investigate the sibling-species composition of An. fluviatilis complex in Kangra valley to ascertain their role in transmission of malaria. The study was undertaken in six villages in Kangra district of Himachal Pradesh, India. A total of 4446 mosquitoes were collected during the one-year study period (2018) and processed in pools of ten for molecular characterization. DNA extraction and multiplex PCR was performed on 900 An. fluviatilis mosquitoes for differentiation of sibling-species. ELISA was used to detect Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in 3790 An. fluviatilis samples. Among prevalent mosquito species, An. fluviatilis was the predominant species constituting 69.5% of total mosquito collection. Sibling-species U was found in 92.22% and species T in 7.78% samples assayed. ELISA confirmed the absence of evidence of malaria parasite in any of the An. fluviatilis mosquitoes screened. Based on the difference in the sequences of conserved regions of the 28SrDNA, sibling-species U was confirmed as prevalent in the study villages. Study revealed that in Kangra district, An. fluviatilis sibling-species U is predominant followed by species T, and both are non-vectors. The absence of malaria parasite and zoophagic nature of An. fluviatilis established through blood meal analysis, confirmed that both U and T are non-vector sibling-species.

Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection’s role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions.