The Leiden Malaria Group developed Modified CRISPR/Cas9 Constructs and Selection Protocol for the Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines
Published in Plos One
The CRISPR/Cas9 protocol we developed provides a simple set of tools to rapidly generate genetically modified P. falciparum lines, in particular transgenic parasites or gene-disruption and gene-mutation mutants that are free of any drug resistance genes. The absence of drug-resistance genes is particularly important in the creation of live genetically attenuated parasites that can be used as a malaria vaccine.
01/1The Leiden Malaria Group published a study demonstrating that Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole
Published in PloS Pathogens
This is the first demonstration of expression of these proteins in the liver and evidence is presented that proteins of one family can transfer phosphatidylcholine in vitro. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.
The Leiden Malaria Group contributed to a study identifying and characterizing a protein essential for the formation of the crystalloid and transmission of the malaria parasite. Published in Proc Natl Acad Sci U S A
We show that formation of the crystalloid - a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony - is dependent on the precisely timed expression of an S-acyl-transferase, an enzyme involved in palmitoylation, a posttranslational modification of proteins (the addition of a C-16 long-chain fatty acid to a cysteine residue).
The Leiden Malaria Group contributed to a genome wide transcriptomic and proteomic analysis of separated male and female sexual cells of malaria parasites. Published in Nucleic Acids Research
This study revealed a large set highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time.
The Leiden Malaria Group contributed to a study showing that the machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites. Published in Nature Communications
Sequestration of red blood cells infected with the human malaria parasite P. falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. In this study two proteins, SBP1 and MAHRP1, are identified that are conserved between rodent and human malaria parasites and that play a role in sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.
The Leiden Malaria Group contributed to the development of a murine model for pre-clinical studies on pathology associated to malaria in pregnancy. Published in Infect. Immun.
Malaria infection during pregnancy leads to abortions, stillbirth, low birth weight and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulphate A (CSA) via var2CSA protein exposed on the IE membrane. P. berghei IE infection in pregnant BALB/c mice is a model for severe PM. We describe a transgenic P. berghei parasite expressing the full length var2CSA extracellular region (domains DBL1X-DBL6ϵ) fused to a P. berghei exported protein (EMAP1) and characterized a var2CSA-based mouse model of placental malaria (PM).
The Malaria Vaccines for the World (MVW)
The Malaria Vaccines for the World (MVW) meeting will take place this year in the LUMC (May 2-4th). This meeting, held once every 3 years, brings together the world’s leading academic and industrial scientists, as well as international regulators, funders and government agencies.
The meeting will describe different aspects of vaccine development, assessment and deployment, as well as funding and regulatory aspects of vaccine implementation and testing. See here for more details.
The Leiden Malaria Group contributed to a study that showed that the multidrug resistance-associated protein 2 (MDR2) is essential for malaria parasite development in the liver (published in Cell Microbiol)
Both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid- to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during malaria parasite liver stage development.
The Leiden Malaria Group contributed to a study that analysed motility of malaria parasites in the skin (published in Elife)
Sporozoite motility and their interaction with dermal blood vessels was analysed using intravital microscopy in mice. Evidence is provided that sporozoites exhibit different types of motility, which is important for successful exit from the inoculation site and invasion of blood vessels.
The Leiden Malaria Group contributed to a study that analysed cytosolic immune responses of host hepatocytes against malaria infection (published in Autophagy)
Both autophagy and nonselective cannonical autophagy events were analysed.
The Leiden Malaria Group contributed to a study that identified two protective liver-stage candidates (published in Sci Rep.)
Chimeric P. berghei parasites expressing the cognate P. falciparum antigen were used to test protective efficacy in mice that were immunised with 10 different P. falciparum vaccine candidate antigens
A Leiden study published in Journal of Experimental Medicine showed that, unexpectedly, malaria parasites can develop without digesting haemoglobin but are restricted to young red blood cells (reticulocytes) for their development and become insensitive to the action of chloroquine.
The Leiden Malaria Group contributed to a study that identified a phospholipase that is involved in disruption of the liver stage parasitophorous vacuole membrane (published in Plos Pathogens)
Dr. Meta Roestenberg, dr. Shahid Khan and prof. Ko Willems van Dijk (Departments of Parasitology and Human Genetics), received a Van de Kamp Fonds-grant (€ 250.000) for research on malaria parasite development in the liver
Dr. Meta Roestenberg received the Gisela Thier Fellowship for research on malaria; specifically to investigate the development of parasites in the liver.
The Leiden Malaria group in collaboration with RadboudUMC published a paper about a genetically modified parasite vaccine (GAP) against malaria, which is now ready for testing in humans (published ineLIFE).
This is an important milestone for malaria research in Leiden and is the culmination of studies that were first initiated here in the mid-90s:
- The Leiden Malaria group was the first group to develop genetic modification in malaria parasites (2 papers published in Science in 1996 and 1997)
- Through analysis of gene-deletion mutants in rodent models of malaria, we discovered that it was possible to create attenuated parasites, which were able to invade the liver but were unable to proceed into the pathogenic blood stage infection. Importantly mice infected (immunised) with these live-attenuated parasites developed protective immunity against an infection with wild type parasites (published in PNAS in 2005).
- In 2008, in the group of Shahid Khan, we started to work on translating these findings into a human vaccine against malaria. This was performed in collaboration with the RadboudUMC (Nijmegen) and the US company Sanaria, and was supported by a grant provided by TI-Pharma. This involved testing and refining many live genetically-attenuated vaccines until we could demonstrate that one met all the necessary pre-clinical safety and protective efficacy standards; it is now ready to advance into testing in humans.
More details on the generation and pre-clinical characterisation of this vaccine can be found in 2 papers published in 2014; one published in the FASEB Journal in May and one that has just appeared in eLIFE.
The Leiden Malaria Group contributed to a study that identified the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis (published in Genome Biology).
Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. Evidence is presented that mRNA of >700 genes is translationally repressed in the female gametocyte which is the precursor cell of the female gamete. These mRNAs are associtated with DOZI and CITH which are translational repressor proteins.
The Leiden Malaria Group recently contributed to a paper entitled ‘A comprehensive evaluation of rodent malaria parasite genomes and gene expression’ (published in BMC Biology)
The consortium that contributed to this study consisted of teams from the Wellcome Trust Sanger Institute (UK), RUMC and LUMC (The Netherlands), NIMR, Univ. of Glasgow and Univ. of Oxford (UK). These analyses resulted in full-length gene models for more than 98% of predicted RMP protein-coding genes. Approximately 60% of these genes have functional annotation, which is comparable to the percentage of functionally annotated genes in the P. falciparum 3D7 reference genome. A high percentage (~90%) of the predicted RMP proteins have orthologs in primate malaria species. This high level of orthology and gene conservation between RMP and primate malaria genomes further supports the use of RMP as experimental models to characterize Plasmodium gene function.
Dr Shahid Khan and Dr Chris Janse of the Leiden Malaria Research Group received a pilot grant of $70,000 from the PATH Malaria Vaccine Initiative (MVI) , an organization established and largely supported by the Bill & Melinda Gates Foundation. This pilot grant has been awarded to examine a novel live genetically-modified malaria-parasite vaccine, which is designed to provide immunity against multiple stages of the malaria parasite. This so called stage-transcending vaccine has been engineered to express malaria-vaccine candidate antigens from different points of the parasite life-cycle in sporozoites, the infectious forms of the parasite that are injected by the mosquito. The Leiden Malaria Research Group are pioneers and leaders in the field of genetic modification of malaria parasites and vaccine research. The pilot study will be first performed in rodents and, if successful, the intention in a second phase to develop this platform for human vaccination.
The Leiden Malaria Group have published a study on Plasmodium mutants expressing Ovalbumin (OVA)(published in Infect. Immunity). They show that the subcellular location of Ovalbumin in Plasmodium berghei blood stages influences the magnitude of T-cell responses.
Dr Shahid Khan and Dr Chris Janse of the Leiden Malaria Research Group received a grant of $340,000 from the PATH Malaria Vaccine Initiative (MVI), an organization established and largely supported by the Bill & Melinda Gates Foundation. The grant has been awarded to develop a number of genetically modified rodent malaria parasites that express proteins from the most virulent human malaria parasite, Plasmodium falciparum. As evaluating malaria vaccine efficacy in humans is difficult, very expensive and highly time consuming these ‘humanized parasites’ will be used in rodents, to rapidly evaluate which vaccines against human malaria-parasite proteins are likely to be most effective in controlling a malaria infection. This research that is being carried out in collaboration with researchers from Seattle BioMed and Johns Hopkins University (both USA) and the best candidates will be directly translated into clinical trials and help inform the final composition of an effective malaria vaccine
The Leiden Malaria Group have published a study identifying and characterizing new members of the malaria parasite's 6-Cys family of proteins (published in FASEB J).
These proteins have critical roles throughout parasite development and are being pursued as targets in anti-malaria vaccination strategies. We show that a number of 6-Cys proteins have critical but distinct roles in the establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development. Specifically, we identify one member (B9) as a potential target to create genetically attenuated parasites suitable for vaccination.
PhD thesis: Jingwen Lin (LUMC, Leiden). Generation of genetically attenuated blood-stage malaria parasites; characterizing growth and virulence in a rodent model of malaria.Read more ....
The Leiden Malaria Research group contributed to a study analysing malaria rhomboid proteases (published in Molecular Microbiology)
Rhomboid-like proteases cleave membrane-anchored proteins within their transmembrane domains. In apicomplexan parasites substrates include molecules that function in parasite motility and host cell invasion. While twoPlasmodium rhomboids, Rhomboid 1 and rhomboid 4, have been examined, the roles of the remaining six rhomboids during the malaria parasite's life cycle are unknown. We present systematic gene deletion analyses of all eight Plasmodium rhomboid-like proteins as a means to discover stage-specific phenotypes and potential functions in the rodent malaria model, P. berghei.
The Leiden Malaria Research group contributed to a study analysing anti-merozoite malaria vaccine candidates using the rodent model Plasmodium berghei (published in Science Reports)
An improved understanding of the mechanisms responsible for protection, or failure of protection, against P. berghei merozoites could guide the development of an efficacious vaccine against P. falciparum.
The Leiden Malaria Research group contributed to a study showing that Hemozoin induces lung inflammation and correlates with Malaria-Associated Acute Respiratory Distress Syndrome (published in Am J Respir Cell Mol Biol.)
Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication and its pathophysiology is insufficiently understood. Both in humans and mouse models, MA-ARDS is characterized by marked pulmonary inflammation. By quantifying hemozoin in the lungs and measuring disease parameters of MA-ARDS, we demonstrate a highly significant correlation between pulmonary hemozoin levels, lung weight, alveolar edema and pulmonary inflammation.
The Leiden Malaria Research group contributed to the generation of fluorescent P. cynomolgi liver stages enabling live imaging and purification of hypnozoite-forms (published in PloS One).
A major challenge for strategies to combat the human malaria parasite P. vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. The generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages enabled live imaging and purification of hypnozoite-forms (hypnozoites)
The Leiden Malaria Research group contributed to a study showing the rodent parasite Plasmodium berghei blood stages export a large and diverse repertoire of proteins into the red blood cell (published in Mol Cell Proteomics).
This study indicates that P. berghei traffics a diverse range of proteins to different cellular locations into the host red blood cell by mechanisms that are analogous to those employed by the human parasite P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.
The Leiden Malaria Research group contributed to a study showing that male gametes of malaria parasites evolve faster than female gametes (published in Evolution, Medicine and Public Health). Read more ..
The Leiden Malaria group published four methods papers in the book: Malaria: Methods and protocols (Methods in Molecular Biology) edited by Robert Menard
- Screening inhibitors of P. berghei blood stages using bioluminescent reporter parasites. Link....
- Quantitative analysis of Plasmodium berghei liver stages by bioluminescence imaging. Link....
- Bioluminescence imaging of P. berghei Schizont sequestration in rodents. Link....
- Bioluminescence imaging of P. berghei Schizont sequestration in rodents. Link....
The Leiden Malaria Research group contributed to a study showing that malaria parasites interfere with the development of immunological memory through expression of an ortholog of macrophage migration inhibitory factor, MIF (published in PNAS)
Malaria parasites interfere with the development of immunological memory through expression of an ortholog of macrophage migration inhibitory factor (MIF). This cytokine enhanced inflammatory cytokine production and also induced antigen-experienced CD4 T cells to develop into short-lived effector cells rather than memory precursor cells.
The Leiden Malaria Research Group and collaborators wrote a review on genetic engineering of attenuated malaria parasites for vaccination (published in Curr. Opin. Biotechnol.).
Vaccination with live-attenuated Plasmodium sporozoites that arrest in the liver can completely protect against a malaria infection both in animal models and in humans. Advances in genetic manipulation of Plasmodium has enabled new approaches to design genetically attenuated parasites (GAPs). Tthe principles in discovery and development of GAPs in preclinical models that are important in selecting GAP parasites for first-in-human clinical studies are dicsussed in this review. The challenges in manufacture, formulation and delivery of a live-attenuated whole parasite malaria vaccine are highlighted, as well as the further refinements that may be implemented in the next generation GAP vaccines.
The Leiden Malaria Research Group and collaborators report on an approach for assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates (published in Vaccine)
The critical first step in the clinical development of a malaria vaccine, based on live-attenuated Plasmodium falciparum sporozoites, is the guarantee of complete arrest in the liver. We report an approach for assessing adequacy of attenuation of genetically attenuated sporozoites in vivo using the Plasmodium berghei model of malaria and P. falciparum sporozoites cultured in primary human hepatocytes.
The Leiden Malaria Research Group contributed to a paper discussing the role of animal models for research into development of new treatments for severe malaria (published in Plos Pathogens)
In light of the recent controversies over the role of animal models for research into the development of new treatments for severe malaria, particularly cerebral disease, a group of scientists came together to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease.
The Leiden Malaria Research Group developed and published a novel method (GIMO transfection) for transgene expression and gene complementation in rodent malaria parasites (published in PlosOne ).
Compared to existing protocols the novel methods of GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation.
The Leiden Malaria Research Group and collaborators showed that CD36-mediated tissue sequestration of malaria parasites is beneficial for growth of the parasites (published in J. Exp. Med) .
These results reveal for the first time the importance of sequestration to a malaria infection, with implications for the development of strategies aimed at reducing pathology by inhibiting tissue sequestration.