We are interested in determining the differences in the transcriptome of select developmental stages of the malaria parasite, Plasmodium.

Malaria remains a major problem in many tropical countries with Plasmodium falciparum accounting for up to 1 million deaths, primarily in infants and children residing in endemic areas of sub-Saharan Africa. P. vivax, the other important species for human malaria is geographically more widespread and causes 80-100 million cases of malaria each year. All the pathology related to malaria is attributed to the blood stage of the parasite life cycle during which Plasmodium merozoites invade and multiply within host erythrocytes. We are interested in understanding the process of RBC invasion by malaria parasites at the molecular level with the goal of blocking their interaction with antibodies to inhibit invasion and kill the parasite. The first generation of recombinant blood stage malaria vaccine candidates based on antigens such as the merozoite surface protein (MSP1) and apical merozoite antigen-1 (AMA01) have been tested in field trials and failed to provide any efficacy against P. falciparum malaria. There is thus an urgent need to identify novel parasite antigens that play a role in invasion and can serve as vaccine candidates that elicit strong antibody responses that block blood stage parasite growth. In this project, we propose to mine the P. falciparum and P. vivax genome databases using bio-informatic tools to identify potential, novel blood stage invasion related parasite antigens that can serve as potential candidates for blood stage malara vaccines. Criteria such as expression profile, presence of conserved domains in orthologs from related plasmodium species, limited polymorphisms in field isolates, interaction with other invasion related proteins and localization to apical organelles or merozoite surface will be used to interrogate P. falciparum and P. vivax genome sequence data and identify potential invasion related antigens that will be selected for validation as vaccine candidates for malaria.

Background: PLA2 is known to regulate vesicle secretion in diverse eukaryotic cells. We are interested in determining the putative role of PLA2 in secretion of apical vesicular organelles called micronemes and rhoptries in P. falciparum merozoites. PLA2 inhibitors such as 4-BPB are known to block growth of the related Apicomplexan parasite Toxoplasma gondii. There are 3 annotated PLA2 genes in the P. falciparum genome database (Pf3D70209100, PF3D71358000, PF3D70924000). We would like to analyze these putative PLA2 genes using bioinformatics to support our drug development studies.    

In Plasmodium falciparum a virulence gene family with 60 var genes codes for the PfEMP1 surface proteins, which undergo antigenic variation. This epigenetically controlled mechanism promotes immune evasion of the parasite. As ncRNAs are emerging as relevant regulators of gene expression we are investigating a GC-rich ncRNA gene family consisting of 15 highly homologous members all positioned next to var gene clusters. We recently found that GC-rich ncRNA transcript associates with the distinct nuclear expression site in which a single var gene is transcribed. We hypothesize that GC-rich ncRNAs interact with the nascent mRNA of var virulence genes. We intend to predict potential RNA-RNA interaction with thermodynamic calculation provided by the RNAup software. In silico prediction of potential binding sites and strength of interaction will help to generate hypotheses and inform our further experimental design.

We are involved, in collboration with the WHO, in the SaMARA which aims at detecting the emergence of antimalarial drug resistance in Africa. Samples (dried blood spots) are collected from the Nantional Malaria Control Programmes in Africa during clinical efficacy studies. My group at the Institut Pasteur in collaboration with the Institut Cochin (Frédéric Ariey) tested these samples and assessed the proportion of parasites harboring SNPs or CNV associated to antimalarial drug resistance. We have previously demonstrated that mutations in the propeller dommain of a Kelch gene located on the chromosome 13 are strongly predictive of artemsinin resistance. Until last year, artemisinin resistance was confined in South east Asia. Recently, we have detected in African samples a high proportion of mutant parasites. This mutation has never been observed before in South east Asia. To perform comprenhensive genomic analysis, we have sequenced (Illumina) all mutants (=24) and paired wild type samples. Analysis of these sequences (and 400 whole genome sequences from parasites collected in Asia, Africa and America, upload on a dedicated website) should allow to: - define if the mutants have emerged locally or have spread from Asia - define the genetic background of these mutants (are specific alleles facilitated the emergence of K13 mutantions) - describe the genomic profile(s) of these mutants (especially the haplotypes associated to multidrug resistance)

In the TSARA project, we aim at developing and validating a molecular platform (Dual Index Targeted Amplicon Deep Sequencing on Illumina iSeq) and a dedicated bioinformatics pipeline for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance. The iSeq Illumina platform, proposed here, is designed for dual indexing of samples, consisting of a 3’ and 5’ individual barcode, which allow the user to connect every sequence generated to a specific sample (up to 384 samples). The development and the validation of this novel molecular tool will be performed at IP Paris first by using DNA extracted from P. falciparum reference strains (3D7, Dd2 and culture-adapted Cambodian strains) (WP1) and second, by using P. falciparum DBS samples collected from two malaria endemic areas (Central African Republic and Cameroon) (WP2). In the WP3, we will host and train staff from IP Bangui and CP Cameroon to this new approach. Our final objective is that Dual Index Targeted Amplicon Deep Sequencing on iSeq along with minimal bioinformatics infrastructure operated in countries that are endemic for malaria to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

In the TSARA project, we aim at developing and validating a molecular platform (Dual Index Targeted Amplicon Deep Sequencing on Illumina iSeq) and a dedicated bioinformatics pipeline for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance. The iSeq Illumina platform, proposed here, is designed for dual indexing of samples, consisting of a 3’ and 5’ individual barcode, which allows the user to connect every sequence generated to a specific sample (up to 384 samples). The development and the validation of this novel molecular tool will be performed at IP Paris first by using DNA extracted from P. falciparum reference strains (3D7, Dd2 and culture-adapted Cambodian strains) (WP1) and second, by using P. falciparum DBS samples collected from two malaria-endemic areas (The central African Republic and Cameroon) (WP2). In the WP3, we will host and train staff from IP Bangui and CP Cameroon to this new approach. Our final objective is that Dual Index Targeted Amplicon Deep Sequencing on iSeq along with minimal bioinformatics infrastructure operated in countries that are endemic for malaria to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure the continued success of the malaria treatment policy

We are working on malaria drug discovery. In an attempt to understand the mode of action of epigenetic inhibitors active against Plasmodium falciparum, we culture the parasite under drug pressure to induce drug resistance, along with a control treated in the same conditions with the solvent only. We then clone resistant and control parasite lines and sequence their genomes to compare them to try to understand which gene is responsible for the resistance and thus may constitute the inhibitor's target (through SNPs, amplification of gene copies).

Malaria remains a major problem in many tropical countries with Plasmodium falciparum accounting for up to 1 million deaths, primarily in infants and children residing in endemic areas of sub-Saharan Africa. P. vivax, the other important species for human malaria is geographically more widespread and causes 80-100 million cases of malaria each year. All the pathology related to malaria is attributed to the blood stage of the parasite life cycle during which Plasmodium merozoites invade and multiply within host erythrocytes. We are interested in understanding the process of RBC invasion by malaria parasites at the molecular level with the goal of blocking their interaction with antibodies to inhibit invasion and kill the parasite. The first generation of recombinant blood stage malaria vaccine candidates based on antigens such as the merozoite surface protein (MSP1) and apical merozoite antigen-1 (AMA01) have been tested in field trials and failed to provide any efficacy against P. falciparum malaria. There is thus an urgent need to identify novel parasite antigens that play a role in invasion and can serve as vaccine candidates that elicit strong antibody responses that block blood stage parasite growth. In this project, we propose to mine the P. falciparum and P. vivax genome databases using bio-informatic tools to identify potential, novel blood stage invasion related parasite antigens that can serve as potential candidates for blood stage malara vaccines. Criteria such as expression profile, presence of conserved domains in orthologs from related plasmodium species, limited polymorphisms in field isolates, interaction with other invasion related proteins and localization to apical organelles or merozoite surface will be used to interrogate P. falciparum and P. vivax genome sequence data and identify potential invasion related antigens that will be selected for validation as vaccine candidates for malaria.

In this project, we aim to identify P. vivax ligands involved in host cell invasion and understand how P. vivax has gained the capacity to infect reticulocytes from Duffy-negative individuals. Our strategy will be based on a 2-step approach by taking full advantage of next-generation sequencing and functional biological analysis (including in vitro invasion assays and infection in humanized mice) and by availing our access to P. vivax isolates from both Duffy-negative and Duffy-positive vivax malaria patients in Madagascar. This strategy should explore and charactere the full repertoire of parasite ligands involved in invasion of human reticulocytes in Malagasy malaria endemic settings where two human populations with distinct Duffy blood group phenotypes (Duffy-negative and Duffy-positive) are frequently exposed to P. vivax. The main objectives of this project are to uncover the molecular basis for the ability of P. vivax to infect reticulocytes from Duffy-positive and Duffy-negative individuals by identifying and defining the role of parasite ligands (including erythrocyte binding proteins (PvEBPs) and reticulocyte binding proteins (PvRBPs)) in mediating invasion and explore the feasibility of targeting combinations of P. vivax invasion related proteins with antibodies to block invasion with high efficiency.