The secreted malarial protein, Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS), is highly conserved among Plasmodium species, and plays a role in the invasion of mosquito midgut cells... Show moreThe secreted malarial protein, Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS), is highly conserved among Plasmodium species, and plays a role in the invasion of mosquito midgut cells and hepatocytes in the vertebrate host. CelTOS was identified as a potential protective antigen based on a proteomic analysis, which showed that CelTOS stimulated significant effector T cells producing IFN-gamma in peripheral blood mononuclear cells (PBMCs) from radiation attenuated sporozoite-immunized, malaria-naive human subjects. In a rodent malaria model, recombinant full-length CelTOS protein/adjuvant combinations induced sterile protection, and in several studies, functional antibodies were produced that had hepatocyte invasion inhibition and transmission-blocking activities. Despite some encouraging results, vaccine approaches using CelTOS will require improvement before it can be considered as an effective vaccine candidate. Here, we report on the use of mRNA vaccine technology to induce humoral and cell-mediated immune responses using this antigen. Several pfceltos encoding mRNA transcripts were assessed for the impact on protein translation levels in vitro. Protein coding sequences included those to evaluate the effects of signal sequence, N-glycosylation on translation, and of nucleoside substitutions. Using in vitro transfection experiments as a pre-screen, we assessed the quality of the expressed CelTOS target relative to the homogeneity, cellular localization, and durability of expression levels. Optimized mRNA transcripts, which demonstrated highest protein expression levels in vitro were selected for encapsulation in lipid nanoparticles (LNP) and used to immunize mice to assess for both humoral and cellular cytokine responses. Our findings indicate that mRNA transcripts encoding pfceltos while potent for inducing antigen-specific cellular cytokine responses in mice, were less able to mount PfCelTOS-specific antibody responses using a two-dose regimen. An additional booster dose was needed to overcome low seroconversion rates in mice. With respect to antibody fine specificities, N-glycosylation site mutated immunogens yielded lower immune responses, particularly to the N-terminus of the molecule. While it remains unclear the impact on CelTOS antigen as immunogen, this study highlights the need to optimize antigen design for vaccine development. Show less
Controlled human infection (CHI) models are an important research tool. Healthy volunteers are experimentally infected with a pathogen. In malaria research the model has been used for decades. Here... Show moreControlled human infection (CHI) models are an important research tool. Healthy volunteers are experimentally infected with a pathogen. In malaria research the model has been used for decades. Here, the model was used to test new Plasmodium falciparum strains, NF135.C10 an NF166.C8, and compare these with the commonly used strain NF54. In addition, a genetically modified malaria vaccine, PfSPZ-GA1, was tested. Unfortunately only few volunteers were protected against malaria.For schistosomiasis, a controlled human schistosomiasis infection (CoHSI) model was developed and hereafter a dose finding study with single-sex male only cercariae was performed. This study showed that in 80% of volunteers 20 cercariae were effective to induce an infection. Although the use of 30 cercariae resulted in 100% infection rate two out of three volunteers developed Katayama syndrome. These side effects were less after infection with 20 cercariae.At last suggestions were made to further improve the CHI model by the use of historical controls. Although this study design can only be used in CHI’s with well-know outcomes these study will be safer by reducing the cumulative risks as less volunteers can be used for these trials. Show less
Background European travellers to endemic countries are at risk of malaria and may be affected by a different range of co-morbidities than natives of endemic regions. The safety profile, especially... Show moreBackground European travellers to endemic countries are at risk of malaria and may be affected by a different range of co-morbidities than natives of endemic regions. The safety profile, especially cardiac issues, of artenimol (previously dihydroartemisinin)-piperaquine (APQ) Eurartesim(R) during treatment of uncomplicated imported falciparum malaria is not adequately described due to the lack of longitudinal studies in this population. The present study was conducted to partially fill this gap. Methods Participants were recruited through Health Care Provider's safety registry in 15 centres across 6 European countries in the period 2013-2016. Adverse events (AE) were collected, with a special focus on cardiovascular safety by including electrocardiogram QT intervals evaluated after correction with either Bazett's (QTcB) or Fridericia's (QTcF) methods, at baseline and after treatment. QTcB and/or QTcF prolongation were defined by a value > 450 ms for males and children and > 470 ms for females. Results Among 294 participants, 30.3% were women, 13.7% of Caucasian origin, 13.5% were current smoker, 13.6% current alcohol consumer and 42.2% declared at least one illness history. The mean (SD) age and body mass index were 39.8 years old (13.2) and 25.9 kg/m(2) (4.7). Among them, 75 reported a total of 129 AE (27 serious), 46 being suspected to be related to APQ (11 serious) and mostly labelled as due to haematological, gastrointestinal, or infection. Women and Non-African participants had significantly (p < 0.05) more AEs. Among AEs, 21 were due to cardiotoxicity (7.1%), mostly QT prolongation, while 6 were due to neurotoxicity (2.0%), mostly dizziness. Using QTcF correction, QT prolongation was observed in 17/143 participants (11.9%), only 2 of them reporting QTcF > 500 ms (milliseconds) but no clinical symptoms. Using QTcB correction increases of > 60 ms were present in 9 participants (6.3%). A trend towards increased prolongation was observed in those over 65 years of age but only a few subjects were in this group. No new safety signal was reported. The overall efficacy rate was 255/257 (99.2%). Conclusions APQ appears as an effective and well-tolerated drug for treatment of malaria in patients recruited in European countries. AEs and QT prolongation were in the range of those obtained in larger cohorts from endemic countries. Trial registration This study has been registered in EU Post-Authorization Studies Register as EUPAS6942 Show less
Miyazaki, Y.; Marin-Mogollon, C.; Imai, T.; Mendes, A.M.; Laak, R. van der; Sturm, A.; ... ; Franke-Fayard, B. 2020
Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in... Show moreChimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP. Show less
Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene... Show moreTransgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. DifferentPlasmodium falciparum(Pf) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standardPfNF54 background and in a recently described CambodianP. falciparumNF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared toPfNF54. We first generatedPfNF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in thePfNF135.C10 line which express GFP driven by thesui1and40spromoters as well as by the previously usedef1 alpha promoter (GFP@ef1 alpha,GFP@sui1, GFP@40s). TheGFP@40sreporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the40spromoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the40spromoter valuable novel tools for analyses ofP. falciparum. Show less
Using a variety of anti-malaria tools has resulted in a steady decline of malaria in several endemic countries worldwide. An effective vaccine will be critical to halt malaria or even succeed to... Show moreUsing a variety of anti-malaria tools has resulted in a steady decline of malaria in several endemic countries worldwide. An effective vaccine will be critical to halt malaria or even succeed to final eradication. In that perspective, we studied the potential of whole sporozoite immunization by bites of P. falciparum infected mosquitoes under chemoprophylaxis (CPS). In this thesis we further explored this CPS model and assessed different immunizing doses, type of chemoprophylaxis and immunological determinants of disease and protection. We found a clear dose dependent efficacy, independent of type of chemoprophylaxis, found CD107a and CD8 T cells producing granzyme B related to protective immunity. In the field many genetically different strains circulate and a future vaccine should be able to cover multiple strains. We re-challenged volunteers with a different strain and found modest heterologously protection.We retrospectively assessed the parasitological dynamics and adverse events using a positive qPCR rather than thick smear and found reduced the clinical symptoms of malaria for volunteers after challenge.Successful malaria eradication will be more likely to be achieved with a multi-disciplinary approach. Additionally, sufficient and continuous funds will proof to be of tremendous necessity. Show less
Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogatemalaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9... Show moreTransgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogatemalaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line mCherry-luc@ etramp10.3). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The mCherry-luc@ etramp10.3 parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology. Show less
For rodent malaria parasites the availability of a variety transgenic parasite lines expressing different reporter proteins under the control of stage-specific or constitutive promoters have been... Show moreFor rodent malaria parasites the availability of a variety transgenic parasite lines expressing different reporter proteins under the control of stage-specific or constitutive promoters have been of great benefit to studies to reveal parasite gene function and to studies focussed on the evaluation of novel drugs and vaccines. The availability of similar transgenic P. falciparum reporter lines would also help to advance gene-function studies and studies aiming at development of new therapies for P. falciparum parasites. In this thesis, we describe a set of studies performed in P. falciparum to develop novel CRISPR/Cas9 methodologies to improve P. falciparum transgenesis and to create novel transgenic parasites that can be used to analyse host-pathogen interactions and for anti-malarial drug and vaccine research. We describe studies aiming at improving CRISPR/Cas9 genetic modification for introduction of transgenes into the genome of P. falciparum using a ‘neutral’ locus, a disruption of the P230p gene using CRISPR/Cas9 showed that P230p like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and the creation of two chimeric P. falciparum parasites (Pf-PvCSP), where the gene encoding circumsporozoite protein (CSP), was replaced by two csp gene variants (VK210 and VK247) showing that species-specific features of CSP govern full sporozoite development. Show less