Transgenic Plasmodium Berghei Parasites Research Articles

A Head-to-Head Comparative Study of the Replication-Competent Vaccinia Virus and AAV1-Based Malaria Vaccine versus RTS,S/AS01 in Murine Models.

Background/Objectives: We developed a multistage Plasmodium falciparum vaccine using a heterologous prime-boost immunization strategy. This involved priming with a highly attenuated, replication-competent vaccinia virus strain LC16m8Δ (m8Δ) and boosting with adeno-associated virus type 1 (AAV1). This approach demonstrated 100% efficacy in both protection and transmission-blocking in a murine model. In this study, we compared our LC16m8∆/AAV1 vaccine, which harbors a gene encoding Pfs25-PfCSP fusion protein, to RTS,S/AS01 (RTS,S) in terms of immune responses, protective efficacy, and transmission-blocking activity (TBA) in murine models. Methods: Mice were immunized following prime-boost vaccine regimens m8∆/AAV1 or RTS,S and challenged with transgenic Plasmodium berghei parasites. Immune responses were assessed via ELISA, and TB efficacy was evaluated using direct feeding assays. Results: m8∆/AAV1 provided complete protection (100%) in BALB/c mice and moderate (40%) protection in C57BL/6 mice, similar to RTS,S. Unlike RTS,S's narrow focus (repeat region), m8∆/AAV1 triggered antibodies for all PfCSP regions (N-terminus, repeat, and C-terminus) with balanced Th1/Th2 ratios. Regarding transmission blockade, serum from m8∆/AAV1-vaccinated BALB/c mice achieved substantial transmission-reducing activity (TRA = 83.02%) and TB activity (TBA = 38.98%)-attributes not observed with RTS,S. Furthermore, m8∆/AAV1 demonstrated durable TB efficacy (94.31% TRA and 63.79% TBA) 100 days post-immunization. Conclusions: These results highlight m8∆/AAV1's dual action in preventing sporozoite invasion and onward transmission, a significant advantage over RTS,S. Consequently, m8∆/AAV1 represents an alternative and a promising vaccine candidate that can enhance malaria control and elimination strategies.

Comparative analyses of functional antibody-mediated inhibition with anti-circumsporozoite monoclonal antibodies against transgenic Plasmodium berghei

BackgroundAcquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays.MethodsIC50 values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 103 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH.ResultsThe IC50 values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC50 of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification.ConclusionsResults from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody IC50 values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system.

Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions.

Virus‐like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP‐based anti‐sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical‐grade material for use in human clinical trials. The R21 construct was re‐engineered to include a C‐tag to allow affinity‐based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C‐tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high‐level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C‐tag platform technologies to enable cGMP‐compliant biomanufacturing of high purity yeast‐expressed VLP‐based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase.

Host cell remodeling is critical for successful Plasmodium replication inside erythrocytes and achieved by targeted export of parasite-encoded proteins. In contrast, during liver infection the malarial parasite appears to avoid protein export, perhaps to limit exposure of parasite antigens by infected liver cells. HSP101, the force-generating ATPase of the protein translocon of exported proteins (PTEX) is the only component that is switched off during early liver infection. Here, we generated transgenic Plasmodium berghei parasite lines that restore liver stage expression of HSP101. HSP101 expression in infected hepatocytes was achieved by swapping the endogenous promoter with the ptex150 promoter and by inserting an additional copy under the control of the elongation one alpha (ef1α) promoter. Both promoters drive constitutive and, hence, also pre-erythrocytic expression. Transgenic parasites were able to complete the life cycle, but failed to export PEXEL-proteins in early liver stages. Our results suggest that PTEX-dependent early liver stage export cannot be restored by addition of HSP101, indicative of alternative export complexes or other functions of the PTEX core complex during liver infection.

Limited Plasmodium sporozoite gliding motility in the absence of TRAP family adhesins

BackgroundPlasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter and cross several cellular and tissue barriers for which they employ a set of surface proteins. Three of these proteins are members of the thrombospondin related anonymous protein (TRAP) family. Here, potential additive, synergistic or antagonistic roles of these adhesion proteins were investigated.MethodsFour transgenic Plasmodium berghei parasite lines that lacked two or all three of the TRAP family adhesins TRAP, TLP and TREP were generated using positive–negative selection. The parasite lines were investigated for their capacity to attach to and move on glass, their ability to egress from oocysts and their capacity to enter mosquito salivary glands. One strain was in addition interrogated for its capacity to infect mice.ResultsThe major phenotype of the TRAP single gene deletion dominates additional gene deletion phenotypes. All parasite lines including the one lacking all three proteins were able to conduct some form of active, if unproductive movement.ConclusionsThe individual TRAP-family adhesins appear to play functionally distinct roles during motility and infection. Other proteins must contribute to substrate adhesion and gliding motility.Graphical

A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of Plasmodium vivax Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic Plasmodium berghei Parasite

Infections with Plasmodium vivax are predominant in the Americas, representing 75% of malaria cases. Previously perceived as benign, malaria vivax is, in fact, a highly debilitating and economically important disease. Considering the high complexity of the malaria parasite life cycle, it has been hypothesized that an effective vaccine formulation against Plasmodium should contain multiple antigens expressed in different parasite stages. Based on that, we analyzed a recombinant P. vivax vaccine formulation mixing the apical membrane antigen 1 ectodomain (PvAMA-1) and a full-length circumsporozoite protein (PvCSP-AllFL) previously studied by our group, which elicits a potent antibody response in mice. Genetically distinct strains of mice (C57BL/6 and BALB/c) were immunized with the proteins, alone or in combination, in the presence of poly(I:C) adjuvant, a TLR3 agonist. In C57BL/6, high-antibody titers were induced against PvAMA-1 and the three PvCSP variants (VK210, VK247, and P. vivax-like). Meanwhile, mixing PvAMA-1 with PvCSP-AllFL had no impact on total IgG antibody titers, which were long-lasting. Moreover, antibodies from immunized mice recognized VK210 sporozoites and blood-stage parasites by immunofluorescence assay. However, in the BALB/c model, the antibody response against PvCSP-AllFL was relatively low. PvAMA-1-specific CD3+CD4+ and CD3+CD8+ T-cell responses were observed in C57BL/6 mice, and the cellular response was impaired by PvCSP-AllFL combination. More relevant, the multistage vaccine formulation provided partial protection in mice challenged with a transgenic Plasmodium berghei sporozoite expressing the homologous PvCSP protein.

Improvement of an in vitro drug selection method for generating transgenic Plasmodium berghei parasites

BackgroundReverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method for generating transgenic parasite of Plasmodium berghei was reported. Using this method, two novel and independent selection markers using the P. berghei heat shock protein 70 promoter was previously established. While the approach permits the easy and flexible genetic manipulation of P. berghei, shortcomings include a low variety in promoter options to drive marker gene expression and increased complexity of the selection procedure. In this study, addressing these issues was attempted.MethodsTo secure a variety of promoters, the use of a P. berghei elongation factor-1α promoter for marker gene expression was attempted. To simplify the procedure of in vitro selection, the establishment of a two cell-cycle culture method and its application for drug selection were attempted.ResultsThe P. berghei elongation factor-1α (pbef-1α) promoter, which is commonly used to drive marker gene expression, was successfully applied as an alternative promoter model for marker gene expression, using the parasite’s codon-optimized marker sequence. To simplify the in vitro selection method, a two cell-cycle culture method in which the merozoite was released by filtration of the culture containing matured schizont-infected erythrocytes was also developed and successfully applied for drug selection.ConclusionThe pbef-1α promoter was successfully applied in an in vitro selection system. The in vitro selection procedure also could be simplified for practical use using a two cell-cycle culture method. These improvements provide a more versatile platform for the genetic manipulation of P. berghei.

In vivo compartmental kinetics of Plasmodium falciparum histidine-rich protein II in the blood of humans and in BALB/c mice infected with a transgenic Plasmodium berghei parasite expressing histidine-rich protein II

BackgroundThe Plasmodium falciparum histidine-rich protein II (PfHRP2) is a common biomarker used in malaria rapid diagnostic tests (RDTs), but can persist in the blood for up to 40 days following curative treatment. The persistence of PfHRP2 presents a false positive limitation to diagnostic interpretation. However, the in vivo dynamics and compartmentalization underlying PfHRP2 persistence have not been fully characterized in the plasma and erythrocyte (RBC) fraction of the whole blood.MethodsThe kinetics and persistence of PfHRP2 in the plasma and RBC fractions of the whole blood were investigated post-treatment in human clinical samples and samples isolated from BALB/c mice infected with a novel transgenic Plasmodium berghei parasite engineered to express PfHRP2 (PbPfHRP2).ResultsPfHRP2 levels in human RBCs were consistently 20–40 times greater than plasma levels, even post-parasite clearance. PfHRP2 positive, DNA negative, once-infected RBCs were identified in patients that comprised 0.1–1% of total RBCs for 6 and 12 days post-treatment, even post-atovaquone–proguanil regimens. Transgenic PbPfHRP2 parasites in BALB/c mice produced and exported tgPfHRP2 to the RBC cytosol similar to P. falciparum. As in humans, tgPfHRP2 levels were found to be approximately 20-fold higher within the RBC fraction than the plasma post-treatment. RBC localized tgPfHRP2 persisted longer than tgPfHRP2 in the plasma after curative treatment. tgPfHRP2 positive, but DNA negative once-infected RBCs were also detected in mouse peripheral blood for 7–9 days after curative treatment.ConclusionsThe data suggest that persistence of PfHRP2 is due to slower clearance of protein from the RBC fraction of the whole blood. This appears to be a result of the presence PfHRP2 in previously infected, pitted cells, as opposed to PfHRP2 binding naïve RBCs in circulation post-treatment. The results thus confirm that the extended duration of RDT positivity after parasite clearance is likely due to pitted, once-infected RBCs that remain positive for PfHRP2.

Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes

BackgroundNon-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic.MethodsA comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qβ phage particle (Qβ-CSP).ResultsMice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qβ-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qβ-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity.ConclusionsDespite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.

The Plasmodium alveolin IMC1a is stabilised by its terminal cysteine motifs and facilitates sporozoite morphogenesis and infectivity in a dose-dependent manner

Apicomplexan parasites possess a unique cortical cytoskeleton structure composed of intermediate filaments. Its building blocks are provided by a conserved family of proteins named alveolins. The core alveolin structure is made up of tandem repeat sequences, thought to be responsible for the filamentous properties of these proteins. A subset of alveolins also possess conserved motifs composed of three closely spaced cysteine residues situated near the ends of the polypeptides. The roles of these cysteine motifs and their contribution to alveolin function remains poorly understood. The sporozoite-expressed IMC1a is unique within the Plasmodium alveolin family in having conserved cysteine motifs at both termini. Using transgenic Plasmodium berghei parasites, we show in this structure-function analysis that mutagenesis of the amino- or carboxy-terminal cysteine motif causes marked reductions in IMC1a protein levels in the parasite, which are accompanied by partial losses of sporozoite shape and infectivity. Our findings give new insight into alveolin function, identifying a dose-dependent effect of alveolin depletion on sporozoite size and infectivity, and vital roles of the terminal cysteine motifs in maintaining alveolin stability in the parasite.

Development of a Plasmodium berghei transgenic parasite expressing the full-length Plasmodium vivax circumsporozoite VK247 protein for testing vaccine efficacy in a murine model.

BackgroundThe approach of using transgenic rodent malaria parasites to assess the immune system’s response to antigenic targets from a human malaria parasite has been shown to be useful for preclinical evaluation of new vaccine formulations. The transgenic Plasmodium berghei parasite line [PvCSP(VK210)/Pb] generated previously expresses the full-length circumsporozoite protein (CSP) VK210 from Plasmodium vivax. The transgenic parasite expresses one of the two most common alleles of CSP, defined by nine amino acids at the central repeat region of this protein. In the present study, a transgenic P. berghei parasite line [PvCSP(VK247)/Pb] expressing the full-length PvCSP(VK247), which is the alternative common allele, was generated and characterized.MethodsThe P. berghei expressing full-length PvCSP(VK247) was generated and examined its applicability to CSP-based vaccine research by examining its biological characteristics in mosquitoes and mice.ResultsSimilar to PvCSP(VK210)/Pb, PvCSP(VK247)/Pb developed normally in mosquitoes and produced infectious sporozoites equipped to generate patent infections in mice. Invasion of HepG2 cells by PvCSP(VK247)/Pb sporozoites was inhibited by an anti-PvCSP(VK247) repeat monoclonal antibody (mAb), but not by an anti-PvCSP(VK210) repeat mAb.ConclusionsThese two transgenic parasites thus far can be used to evaluate the potential efficacy of PvCSP-based vaccine candidates encompassing the two major genetic variants in preclinical trials.

The Plasmodium translocon of exported proteins component EXP2 is critical for establishing a patent malaria infection in mice.

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium translocon of exported proteins (PTEX) and a cleavable Plasmodium export element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic Plasmodium berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection.

Implications of Glutathione Levels in the Plasmodium berghei Response to Chloroquine and Artemisinin.

Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ) and artemisinin (ART) are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ) and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT) and the multidrug resistant gene (pfmdr), CQ resistance has also been associated with increased parasite glutathione (GSH) levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs) encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko) or higher (pbggcs-oe) levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ). Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.

Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly

Malaria parasites possess unique subcellular structures and organelles. One of these is the crystalloid, a multivesicular organelle that forms during the parasite’s development in vector mosquitoes. The formation and function of these organelles remain poorly understood. A family of six conserved and modular proteins named LCCL-lectin adhesive-like proteins (LAPs), which have essential roles in sporozoite transmission, localise to the crystalloids. In this study we analyse crystalloid formation using transgenic Plasmodium berghei parasites expressing GFP-tagged LAP3. We show that deletion of the LCCL domain from LAP3 causes retarded crystalloid development, while knockout of LAP3 prevents formation of the organelle. Our data reveal that the process of crystalloid formation involves active relocation of endoplasmic reticulum-derived vesicles to common assembly points via microtubule-dependent transport. Inhibition of microtubule-dependent cargo transport disrupts this process and replicates the LCCL domain deletion mutant phenotype in wildtype parasites. These findings provide the first clear insight into crystalloid biogenesis, demonstrating a fundamental role for the LAP family in this process, and identifying the crystalloid and its formation as potential targets for malaria transmission control.

Assessment of the prophylactic activity and pharmacokinetic profile of oral tafenoquine compared to primaquine for inhibition of liver stage malaria infections

BackgroundAs anti-malarial drug resistance escalates, new safe and effective medications are necessary to prevent and treat malaria infections. The US Army is developing tafenoquine (TQ), an analogue of primaquine (PQ), which is expected to be more effective in preventing malaria in deployed military personnel.MethodsTo compare the prophylactic efficacy of TQ and PQ, a transgenic Plasmodium berghei parasite expressing the bioluminescent reporter protein luciferase was utilized to visualize and quantify parasite development in C57BL/6 albino mice treated with PQ and TQ in single or multiple regimens using a real-time in vivo imaging system (IVIS). As an additional endpoint, blood stage parasitaemia was monitored by flow cytometry. Comparative pharmacokinetic (PK) and liver distribution studies of oral and intravenous PQ and TQ were also performed.ResultsMice treated orally with three doses of TQ at 5 mg/kg three doses of PQ at 25 mg/kg demonstrated no bioluminescence liver signal and no blood stage parasitaemia was observed suggesting both drugs showed 100% causal activity at the doses tested. Single dose oral treatment with 5 mg TQ or 25 mg of PQ, however, yielded different results as only TQ treatment resulted in causal prophylaxis in P. berghei sporozoite-infected mice. TQ is highly effective for causal prophylaxis in mice at a minimal curative single oral dose of 5 mg/kg, which is a five-fold improvement in potency versus PQ. PK studies of the two drugs administered orally to mice showed that the absolute bioavailability of oral TQ was 3.5-fold higher than PQ, and the AUC of oral TQ was 94-fold higher than oral PQ. The elimination half-life of oral TQ in mice was 28 times longer than PQ, and the liver tissue distribution of TQ revealed an AUC that was 188-fold higher than PQ.ConclusionsThe increased drug exposure levels and longer exposure time of oral TQ in the plasma and livers of mice highlight the lead quality attributes that explain the much improved efficacy of TQ when compared to PQ.

Efficacy of a Plasmodium vivax malaria vaccine using ChAd63 and modified vaccinia Ankara expressing thrombospondin-related anonymous protein as assessed with transgenic Plasmodium berghei parasites.

Plasmodium vivax is the world's most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8+ T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application.

4(1H)-Quinolones with liver stage activity against Plasmodium berghei.

With the exception of primaquine, tafenoquine, and atovaquone, there are very few antimalarials that target liver stage parasites. In this study, a transgenic Plasmodium berghei parasite (1052Cl1; PbGFP-Luc(con)) that expresses luciferase was used to assess the anti-liver stage parasite activity of ICI 56,780, a 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), as well as two 3-phenyl-4(1H)-quinolones (P4Q), P4Q-146 and P4Q-158, by using bioluminescent imaging (BLI). Results showed that all of the compounds were active against liver stage parasites; however, ICI 56,780 and P4Q-158 were the most active, with low nanomolar activity in vitro and causal prophylactic activity in vivo. This potent activity makes these compounds ideal candidates for advancement as novel antimalarials.

Evaluation of immunity against malaria using luciferase-expressing Plasmodium berghei parasites

BackgroundMeasurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic Plasmodium berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter luciferase. This study explores the benefit of using these parasites for the evaluation of immunity against malaria, compared to qRT-PCR techniques in vivo and in vitro.MethodsMice were immunized with either radiation attenuated sporozoites (RAS) or wildtype sporozoites under chloroquine prophylaxis (CPS) and challenged with PbGFP-Luccon. The in vitro transgenic sporozoites neutralization assay (TSNA) was adapted by replacing PbCS(Pf) parasites for PbGFP-Luccon parasites.ResultsApplication of PbGFP-Luccon transgenic parasites provides live quantitative visual information about the relation between parasite liver load and protection. Moreover, fast and reproducible results are obtained by using these parasites in the transgenic sporozoites neutralization assay, measuring functional antibody-mediated immune responses.ConclusionsPbGFP-Luccon parasites are a straightforward and valuable tool for comprehension of the biological and immunological principles underlying protection against malaria.

Structure–Function Analysis of Malaria Proteins by Gene Targeting – A Response

We have generated transgenic Plasmodium berghei parasites in which the endogenous gene encoding thrombospondin-related anonymous protein (TRAP) was replaced by either wild-type (WT) P. falciparum TRAP (PfTRAP) or by mutated versions of PfTRAP carrying amino acids substitutions or deletions in the A domain or in the thrombospondin-related motif1. This strategy was employed in a structure–function analysis, which aimed to circumvent limitations due to the availability of only one selectable marker to transform P. berghei. We did not attempt to modify the endogenous gene encoding TRAP by using an insertion-targeting approach because we were (and still are) convinced that genetic reversion is a limitation in structure–function studies. A reversion frequency of 1% should not be underestimated. If the revertant parasites were 50–100% more efficient than the mutated ones in infecting target cells or in gliding motility, their presence would confound the phenotypic analysis of the mutated parasites.