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Effects of a double-edged sword: exploiting the interaction between immunity and chemotherapy in murine and ovine models of congenital neosporosis and toxoplasmosis

English title Effects of a double-edged sword: exploiting the interaction between immunity and chemotherapy in murine and ovine models of congenital neosporosis and toxoplasmosis
Applicant Hemphill Andrew
Number 184662
Funding scheme Project funding (Div. I-III)
Research institution Institut für Parasitologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Immunology, Immunopathology
Start/End 01.04.2019 - 31.03.2023
Approved amount 846'720.00
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All Disciplines (2)

Discipline
Immunology, Immunopathology
Veterinary Medicine

Keywords (13)

Toxoplasma gondii; pregnant ruminant model; fetal loss; Neospora caninum; quinolones; vaccination; chemotherapy; calcium dependent kinase inhibitors; vaccine-linked chemotherapy; recombinant antigens; Apicomplexa; pregnant mouse model; abortion

Lay Summary (German)

Lead
Dieses Projekt untersucht den Aspekt der Entwicklung von Wirkstofftoleranz und Persistenz bei Toxoplasma gondii und Neospora caninum, zwei wichtigen Aborterregern bei Mensch (Toxoplasma) und Tier (Toxoplasma, Neospora)
Lay summary

Neospora caninum and Toxoplasma gondii gehören zu den Apikomplexa, und sind wichtige Krankheitserreger in Haus- und Nutztierenudn von grosser ökonomischen Bedeutung. Toxoplasma ist auch für Menschen infektiös. Es gibt bisher keine Subunit-Vakzine welche die Infektion verhindert. Der Einsatz von Wirkstoffen könnte eine wichtige Alternative, oder Komplementierung, von Vakzine-basierender Präventionsstrategien sein.  Wir haben einige Wirkstoffe identifiziert, welche in trächtigen Maus- und Schafmodellen der Neosporose und Toxoplasmose sehr effektiv Aborte verhindern, welche aber in der in vitro Kultur nicht parasitozid wirken. Die Parasiten zeigen ein äusserst grosses Adaptationspotential und werden Wirkstoff-tolerant. Dies impliziert, dass Wirkstoffe in Parasiten und/oder Wirtszellen Veränderungen hervorrufen, welche in vivo eine protektive Immunität initiieren. Welche Mechanismen bei diesen Vorgängen eine Rolle spielen, und wie sich Wirkstofftoleranz und/oder -persistenz bei den Apikomplexa entwickelt, ist nicht bekannt. Wir verwenden vergleichende Proteomics um Proteine zu identifizieren, die in Wirkstoff-toleranten Parasiten hochreguliert sind. Die Funktion und die Validierung dieser Proteine als Wirkstoff- und Vakzine-Targets, wird genauer untersucht. Basierend auf spezifischen Antigenen soll ein kombinierter Vakzine / Chemotherapie Approach zur Behandlung/Prävention von Neosporose und Toxoplasmose etabliert werden. 

Direct link to Lay Summary Last update: 01.04.2019

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Associated projects

Number Title Start Funding scheme
146162 Of mice and sheep: prevention and treatment of Neospora caninum infections 01.04.2013 Project funding (Div. I-III)
173718 Ruthenium Complexes for the Treatment of Protozoan Diseases of Medical and Veterinary Importance 01.09.2017 Sinergia
165782 The cross-talk between chemotherapy and immunity in murine and ovine neosporosis disease 01.04.2016 Project funding (Div. I-III)

Abstract

Background: Apicomplexan parasites that cause major diseases in farm animals such as Neospora caninum and Toxoplasma gondii have a tremendous global economic impact. Despite intensive research, efficient subunit vaccines preventing infection have not been developed to date. The use of drugs that limit parasite dissemination could offer an alternative and/or complementation to vaccination. Bumped kinase inhibitors (BKIs) of calcium-dependent protein kinase 1, and quinolones such as buparvaquone (BPQ) and endochin-like quinolones (ELQs), which interfere in oxidative phosphorylation in the mitochondrion, strongly inhibit these parasites in 3-4-day proliferation assays that are used to determine IC50 values in vitro. However, we observed that these parasites develop tolerance to these otherwise highly efficacious compounds upon long-term exposure, thus resuming proliferation, albeit at a lower rate, even in the presence of elevated drug levels. Nevertheless, BKIs, BPQ and ELQs were shown to be highly efficacious in reducing vertical transmission and fetal loss due to neosporosis/toxoplasmosis in pregnant mouse and sheep models, and appeared efficacious in suppressing parasite growth many weeks after treatment. This suggests that these drugs, even though they do not act parasiticidal per se, induce modifications in the parasite or parasite-infected host cells in such a way that the immune response elicited during infection and treatment can prevent disease in the long term. However, the basis for this adaptive potential, and how immunity tackles these parasites during drug treatment, has not been studied so far. That is what this project is about. Working hypotheses: 1.Neospora and Toxoplasma can adapt to a variety of drugs and develop long-term drug tolerance. 2.Drug tolerance is based on, or at least associated with, changes in gene expression and proteome composition.3.Drugs not acting parasiticidal, induce parasite and/or host cell changes that elicit protective immunity.4.The features of this protective immunity can be exploited to develop efficacious vaccine-linked chemotherapy approaches.Specific aims:1.Generation and characterization of drug-adapted N. caninum and T. gondii strains in vitro.2.Identification and detailed characterization of up- and down-regulated proteins expressed in drug-adapted versus non-adapted tachyzoites, or respective host cells, with a focus on novel potential vaccine candidates, or immune receptors.3.Assessment of early and late immune responses in drug-treated versus non-drug-treated pregnant mouse models, 4.Evaluation of combined vaccination/drug treatment approaches in pregnant murine and ovine infection models.Experimental design and/or methods: 1: Long term tachyzoite in vitro culture to develop strains adapted to elevated drug concentrations. Characterization of stability of tolerance phenotype, invasion and proliferation, drug susceptibility, sequence of the respective drug target genes, cross-adaptation to other drugs, and virulence in mice.2: Comparative proteomic analysis by LC-MS in drug-adapted versus non-adapted strains. Identification of upregulated proteins with vaccine potential by bioinformatics tools. Production of respective immunogenic antigens in E. coli, purification and assessments with sera from drug-treated and non-treated infected mice. Localization and monitoring of protein expression, and specific transcript quantification by real time PCR. Functional characterization of differentially regulated proteins by generating knockout strains using CRISPR-Cas9, studies on the ability of these lines to develop drug tolerance, and virulence assessment in mice. 3: Comparative assessment of early (8 d.p.i) and late (44 d.p.i.) immune parameters in drug-treated and non-treated infected dams and pups, including clinical signs, immuno-histology, serology and quantitative real time PCR for determination of parasite load. 4: Evaluation of a vaccine-linked drug treatment approach (vaccination-infection-treatment) employing already established and novel vaccine candidates in pregnant murine and ovine infection models, assessment of humoral and cellular immune responses in dams and offspring, efficacy and clinical parameters. Expected value of the project: We will analyze the basic biological features of drug-adapted Neospora and Toxoplasma tachyzoites, and the gene expression changes that occur during the development of drug tolerance. We will study immune parameters during infection and treatment. In addition, we will investigate how this knowledge could be possibly exploited for designing novel vaccine-linked chemotherapy approaches against neosporosis and toxoplasmosis, employing established and standardized murine and ovine infection models. The development of drug-tolerant phenotypes has been widely studied in bacteria, and combining vaccines with drug treatment has been shown to increase the efficacy of treatments in cancer, while these aspects have not been studied in cyst-forming apicomplexans before.
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