Nathalie Moiré

Treatment of congenital toxoplasmosis is potentially toxic and above all too expensive to be administered systematically in middle-income countries such as Morocco. There is therefore a real interest in discovering alternative treatments that would be financially accessible to all. In this context, plants used in traditional medicine and purchased in markets are good candidates. Aqueous extract of Ammi visnaga seeds had no inhibitory effect against T. gondii tachyzoites intracellular growth in vitro and induced a cytotoxic effect on host cells. In contrast, ethanolic extract of A. visnaga seeds showed anti- Toxoplasma effect with low cytotoxicity, indicating the compounds extracted differed according to the solvent used. Aqueous extracts of Punica granatum peel and Syzygium aromaticum flower buds also showed anti- Toxoplasma effect with low cytotoxicity. All the extracts tested, with a greater effect from aqueous and ethanolic extracts of A. visnaga seeds, induced apoptosis-like of extracellular tachyzoites, as determined by exposure of phosphatidylserine on tachyzoite surface and DNA fragmentation. Finally, the aqueous extracts of A. visnaga seeds, P. granatum peel and S. aromaticum flower buds exhibited antioxidant properties. Phytochemical analysis indicated that coumarins and sterols from the aqueous extract of A. visnaga , saponins from the ethanolic extract of A. visnaga , gallic tannins from the extract of P. granatum , and phenols from the extract of S. aromaticum were certainly the main components responsible for the different effects observed. The results suggested that the seeds of A. visnaga and the peel of P. granatum are the two best candidates for possible preclinical studies on toxoplasmosis.

Toxoplasma gondii holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether T. gondii specificity can be improved by surface display of scFv directed against dendritic cells’ endocytic receptor, DEC205, and immune checkpoint PD-L1. Anti-DEC205 scFv was anchored to the T. gondii surface either directly via glycosylphosphatidylinositol (GPI) or by fusion with the SAG1 protein. Both constructs were successfully expressed, but the binding results suggested that the anti-DEC-SAG1 scFv had more reliable functionality towards recombinant DEC protein and DEC205-expressing MutuDC cells. Two anti-PD-L1 scFv constructs were developed that differed in the localization of the HA tag. Both constructs were adequately expressed, but the localization of the HA tag determined the functionality by binding to PD-L1 protein. Co-incubation of T. gondii displaying anti-PD-L1 scFv with tumor cells expressing/displaying different levels of PD-L1 showed strong binding depending on the level of available biomarker. Neutralization assays confirmed that binding was due to the specific interaction between anti-PD-L1 scFv and its ligand. A mixed-cell assay showed that T. gondii expressing anti-PD-L1 scFv predominately targets the PD-L1-positive cells, with negligible off-target binding. The recombinant RH-PD-L1-C strain showed increased killing ability on PD-L1+ tumor cell lines compared to the parental strain. Moreover, a co-culture assay of target tumor cells and effector CD8+ T cells showed that our model could inhibit PD1/PD-L1 interaction and potentiate T-cell immune response. These findings highlight surface display of antibody fragments as a promising strategy of targeting replicative T. gondii strains while minimizing nonspecific binding.

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

Vaccination with the live attenuatedToxoplasma gondiiMic1.3KO strain induced long-lasting immunity against challenge withToxoplasma gondiitype I and type II strains. The involvement of regulatory T cells (Tregs) in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4⁺Foxp3⁺T regulatory cells followed by recruitment/expansion of CD4⁺Foxp3⁻CD25⁺effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Tregs were of the TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4⁺Foxp3⁺cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of the TH1/TH17-like type. In addition,in vivoTreg induction in RH-infected mice with interleukin-2 (IL-2)/anti-IL-2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Tregs may contribute to the protective response after vaccination with Mic1.3KO.