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Of mice and sheep: prevention and treatment of Neospora caninum infections

Applicant Hemphill Andrew
Number 146162
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 01.04.2013 - 31.03.2016
Approved amount 493'920.00
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All Disciplines (2)

Discipline
Immunology, Immunopathology
Veterinary Medicine

Keywords (9)

Neospora caninum; recombinant antigens; rhoptries; calcium dependent protein kinase inhibitors; invasion; chemotherapy; micronemes; vaccine; Apicomplexa

Lay Summary (German)

Lead
Neospora caninum, ein weltweit verbreiteter Aborterreger und Verursacher von neuromuskulären Störungen bei Rindern und Hunden, lebt unbemerkt in chronisch infizierten Tieren und wird während der Trächtigkeit transplazental auf die Nachkommen übertragen. Das Mausmodell ist ein häufig verwendetes Infektionsmodell, aber aufgrund der immunologischen Unterschiede zwischen Rindern und Mäusen ist dessen Relevanz grundsätzlich in Frage zu stellen.
Lay summary

Inhalt und Ziel des Forschungsprojektes

Ziel des Projektes ist, neue Massnahmen zur Prävention und Therapie der Neosporose zu entwickeln. Targets sind Proteine, die an der Wirtszellinvasion beteiligt sind. Konkret werden folgende Aspekte untersucht: (i) Expression von rekombinanten Antigenen in Fusion mit OprI –Lipoprotein und Evaluation dieser Antigene als Vakzine im Mausmodell; (ii) Evaluation von Inhibitoren von Ca2+-abhängigen Protein Kinasen, welche wichtig sind für die Wirtszellinvasion; (iii) Etablierung von Schafen als experimentelles Modell zur Untersuchung von Vakzinen und Chemotherapeutika.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojektes

Das Projekt legt die Grundlagen für die Entwicklung von neuen Vakzinen und chemotherapeutischen Ansätzen zur Bekämpfung der Neosporose. Die Etablierung eines Modelles in Schafen ist ein wichtiger Schritt in Richtung eines realistischeren experimentellen Modells welches die Situation in Rindern besser simuliert als das Mausmodell. Dank der Identifizierung von Impf- und Wirkstoffen erhalten wir Einblicke in die komplexe Biologie intrazellulärer Parasiten und Wirts-Parasit Interaktionen. Prospektiv ist dadurch ein Erkenntnisgewinn zu erwarten, der auch für andere Parasitosen relevant ist. 


Direct link to Lay Summary Last update: 13.05.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Approaches for the vaccination and treatment of Neospora caninum infections in mice and ruminant models.
Hemphill Andrew, Aguado-Martínez Adriana, Müller Joachim (2016), Approaches for the vaccination and treatment of Neospora caninum infections in mice and ruminant models., in Parasitology, 143(3), 245-59.
Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.
Pastor-Fernández Iván, Regidor-Cerrillo Javier, Jiménez-Ruiz Elena, Álvarez-García Gema, Marugán-Hernández Virginia, Hemphill Andrew, Ortega-Mora Luis M (2016), Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle., in Parasitology, 143(1), 97-113.
In vitro screening of the open source MMV malaria box reveals novel compounds with profound activities against Theileria annulata schizonts.
Hostettler Isabel, Müller Joachim, Hemphill Andrew (2016), In vitro screening of the open source MMV malaria box reveals novel compounds with profound activities against Theileria annulata schizonts., in Antimicrobial agents and chemotherapy, 1.
Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model.
Müller Joachim, Aguado-Martínez Adriana, Manser Vera, Wong Ho Ning, Haynes Richard K, Hemphill Andrew (2016), Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model., in Veterinary research, 47, 32-32.
Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation.
Arranz-Solís David, Benavides Julio, Regidor-Cerrillo Javier, Horcajo Pilar, Castaño Pablo, del Carmen Ferreras María, Jiménez-Pelayo Laura, Collantes-Fernández Esther, Ferre Ignacio, Hemphill Andrew, Pérez Valentín, Ortega-Mora Luis Miguel (2016), Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation., in Veterinary research, 47, 2-2.
A live vaccine against Neospora caninum abortions in cattle.
Reichel Michael P, Moore Dadín P, Hemphill Andrew, Ortega-Mora Luis M, Dubey J P, Ellis John T (2015), A live vaccine against Neospora caninum abortions in cattle., in Vaccine, 33(11), 1299-301.
Buparvaquone is active against Neospora caninum in vitro and in experimentally infected mice.
Müller Joachim, Aguado-Martinez Adriana, Manser Vera, Balmer Vreni, Winzer Pablo, Ritler Dominic, Hostettler Isabel, Arranz-Solís David, Ortega-Mora Luis, Hemphill Andrew (2015), Buparvaquone is active against Neospora caninum in vitro and in experimentally infected mice., in International journal for parasitology. Drugs and drug resistance, 5(1), 16-25.
Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model.
Arranz-Solís David, Aguado-Martínez Adriana, Müller Joachim, Regidor-Cerrillo Javier, Ortega-Mora Luis Miguel, Hemphill Andrew (2015), Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model., in Veterinary parasitology, 211(3-4), 133-40.
In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.
Winzer Pablo, Müller Joachim, Aguado-Martínez Adriana, Rahman Mahbubur, Balmer Vreni, Manser Vera, Ortega-Mora Luis Miguel, Ojo Kayode K, Fan Erkang, Maly Dustin J, Van Voorhis Wesley C, Hemphill Andrew (2015), In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum., in Antimicrobial agents and chemotherapy, 59(10), 6361-74.
In vitro effects of new artemisinin derivatives in Neospora caninum-infected human fibroblasts.
Müller Joachim, Balmer Vreni, Winzer Pablo, Rahman Mahbubur, Manser Vera, Haynes Richard K, Hemphill Andrew (2015), In vitro effects of new artemisinin derivatives in Neospora caninum-infected human fibroblasts., in International journal of antimicrobial agents, 46(1), 88-93.
Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis.
Arranz-Solís David, Benavides Julio, Regidor-Cerrillo Javier, Fuertes Miguel, Ferre Ignacio, Ferreras Maria Del Carmen, Collantes-Fernández Esther, Hemphill Andrew, Pérez Valentín, Ortega-Mora Luis Miguel (2015), Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis., in Veterinary research, 46, 19-19.
A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.
Hostettler Isabel, Müller Joachim, Stephens Chad E, Haynes Richard, Hemphill Andrew (2014), A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts., in International journal for parasitology. Drugs and drug resistance, 4(3), 201-9.
A review on bovine besnoitiosis: a disease with economic impact in herd health management, caused by Besnoitia besnoiti (Franco and Borges, ).
Cortes Helder, Leitão Alexandre, Gottstein Bruno, Hemphill Andrew (2014), A review on bovine besnoitiosis: a disease with economic impact in herd health management, caused by Besnoitia besnoiti (Franco and Borges, )., in Parasitology, 141(11), 1406-17.
A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis.
Pastor-Fernández Iván, Arranz-Solís David, Regidor-Cerrillo Javier, Álvarez-García Gema, Hemphill Andrew, García-Culebras Alicia, Cuevas-Martín Carmen, Ortega-Mora Luis M (2014), A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis., in Veterinary parasitology, 207(3-4), 203-15.
ApiCOWplexa 2013--2nd International Meeting on Apicomplexan Parasites in Farm Animals.
Hemphill Andrew, Leitão Alexandre (2014), ApiCOWplexa 2013--2nd International Meeting on Apicomplexan Parasites in Farm Animals., in Parasitology, 141(11), 1355-8.
Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.
Ojo Kayode K, Reid Molly C, Kallur Siddaramaiah Latha, Müller Joachim, Winzer Pablo, Zhang Zhongsheng, Keyloun Katelyn R, Vidadala Rama Subba Rao, Merritt Ethan A, Hol Wim G J, Maly Dustin J, Fan Erkang, Van Voorhis Wesley C, Hemphill Andrew (2014), Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy., in PloS one, 9(3), 92929-92929.
Phenotypic and molecular characterization of hyperpigmented group B Streptococci.
Lupo Agnese, Ruppen Corinne, Hemphill Andrew, Spellerberg Barbara, Sendi Parham (2014), Phenotypic and molecular characterization of hyperpigmented group B Streptococci., in International journal of medical microbiology : IJMM, 304(5-6), 717-24.
Vaccines against neosporosis: what can we learn from the past studies?
Monney Thierry, Hemphill Andrew (2014), Vaccines against neosporosis: what can we learn from the past studies?, in Experimental parasitology, 140, 52-70.
Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).
Spekker K, Leineweber M, Degrandi D, Ince V, Brunder S, Schmidt S K, Stuhlsatz S, Howard J C, Schares G, Degistirici O, Meisel R, Sorg R V, Seissler J, Hemphill A, Pfeffer K, Däubener W (2013), Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs)., in Medical microbiology and immunology, 202(3), 197-206.
Differential effects of intranasal vaccination with recombinant NcPDI in different mouse models of Neospora caninum infection.
Debache K, Hemphill A (2013), Differential effects of intranasal vaccination with recombinant NcPDI in different mouse models of Neospora caninum infection., in Parasite immunology, 35(1), 11-20.
In vitro culture systems for the study of apicomplexan parasites in farm animals.
Müller Joachim, Hemphill Andrew (2013), In vitro culture systems for the study of apicomplexan parasites in farm animals., in International journal for parasitology, 43(2), 115-24.
In vitro effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites.
Barna Fabienne, Debache Karim, Vock Carsten A, Küster Tatiana, Hemphill Andrew (2013), In vitro effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites., in Antimicrobial agents and chemotherapy, 57(11), 5747-54.
Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro.
Alaeddine Ferial, Hemphill Andrew, Debache Karim, Guionaud Christophe (2013), Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro., in Parasitology, 140(8), 1033-50.
New approaches for the identification of drug targets in protozoan parasites.
Müller Joachim, Hemphill Andrew (2013), New approaches for the identification of drug targets in protozoan parasites., in International review of cell and molecular biology, 301, 359-401.
Novel amidines and analogues as promising agents against intracellular parasites: a systematic review.
Soeiro M N C, Werbovetz K, Boykin D W, Wilson W D, Wang M Z, Hemphill A (2013), Novel amidines and analogues as promising agents against intracellular parasites: a systematic review., in Parasitology, 140(8), 929-51.
Proteins mediating the Neospora caninum-host cell interaction as targets for vaccination.
Hemphill Andrew, Debache Karim, Monney Thierry, Schorer Michelle, Guionaud Christophe, Alaeddine Ferial, Mueller Norbert, Mueller Joachim (2013), Proteins mediating the Neospora caninum-host cell interaction as targets for vaccination., in Frontiers in bioscience (Elite edition), 5, 23-36.
Use of a Th1 Stimulator Adjuvant for Vaccination against Neospora caninum Infection in the Pregnant Mouse Model.
Monney Thierry, Grandgirard Denis, Leib Stephen L, Hemphill Andrew (2013), Use of a Th1 Stimulator Adjuvant for Vaccination against Neospora caninum Infection in the Pregnant Mouse Model., in Pathogens (Basel, Switzerland), 2(2), 193-208.

Collaboration

Group / person Country
Types of collaboration
Prof. Luis Ortega-Mora, Universidad Complutense, Madrid Spain (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Richard Haynes South Africa (Africa)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Profs. Bruno Gottstein / Norbert Müller, Institute of Parasitology, Universität Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
PD Dr. Joachim Müller, Institute of Parasitology, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Wes van Voorhis, Dr. Kajode Ojo, Dept of Medicine, Univ of Washington United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Alexandre Leitao, University of Lisbon Portugal (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Stephen Leib, Labor Spiez & Institute for Infectious Diseases (IFIK), Universität Bern Switzerland (Europe)
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
27th meeting of the German Society for Parasitology Talk given at a conference Repurposing of antiparasitic drugs – the hydroxy-napthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model 09.03.2016 Göttingen, Germany Aguado-Martinez Adriana; Hemphill Andrew; Hostettler Isabel;
VII. European Congress of Protistology Talk given at a conference Apicomplexan parasites: survival artists and their astonishing adaptive potential to anti- proliferative drugs 07.09.2015 Sevilla, Spain Hemphill Andrew;
Apicowplexa 2015 Talk given at a conference Dose titration of the Neospora caninum virulent Nc-Spain7 isolate in a pregnant mouse model 30.06.2015 Edinburgh, Great Britain and Northern Ireland Hemphill Andrew; Aguado-Martinez Adriana;
Apicowplexa 2015 Talk given at a conference Development and application of a novel quantitative reverse-transcriptase PCR assay to identify drugs targeting intracellular Theileria annulata schizonts 30.06.2015 Edinburgh, Great Britain and Northern Ireland Hostettler Isabel; Hemphill Andrew;
Apicowplexa 2015 Talk given at a conference A new bacterial lipoprotein-based chimeric vaccine induces a mixed Th1/Th2 cytokine response but fails to protect against N. caninum challenge in pregnant mice 30.06.2015 Edinbourgh, Great Britain and Northern Ireland Aguado-Martinez Adriana; Hemphill Andrew; Balmer Verena;
16th Drug Design & Development Seminar,Berlin Talk given at a conference Effects of the bumped kinase inhibitor 1294 in the related cyst-forming apicomplexans Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. 16.03.2015 Berlin, Germany Hemphill Andrew;
GCB Symposium Bern Talk given at a conference Development and application of a novel quantitative reverse-transcriptase PCR assay to identify drugs targeting intracellular Theileria annulata schizonts 28.01.2015 Bern, Switzerland Hostettler Isabel;
Seminar, University of Evora, Portugal Individual talk Vaccines and drugs against Neospora caninum and related apicomplexans 19.11.2014 Evora, Portugal Hemphill Andrew;
11th International Coccidiosis Conference, Dresden Germany Talk given at a conference Targeting host cell invasion mechanisms for the development of drugs and vaccines against Neospora caninum infection – a valid approach? 28.09.2014 Dresden, Germany Hemphill Andrew;
Joint meeting DGP and SGTP Talk given at a conference Use of a quantitative real time RT-PCR assay to detect drug activities against intracellular Theileria annulata schizonts 16.07.2014 Zürich, Switzerland Hostettler Isabel;
GCB Symposium Poster Quantitative real-time RT- PCR as a tool for drug screening in Theileria infected cells 29.01.2014 Bern, Switzerland Hemphill Andrew;
Apicowplexa 2013 Talk given at a conference In vitro (& in vivo) effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites. 31.10.2013 Kusadasi, Turkey Balmer Verena; Hemphill Andrew;
24th International Conference of the World Association for the Advancement of Veterinary Parasitology Talk given at a conference In vitro (& in vivo) effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites 25.08.2013 Perth, Australia Hemphill Andrew;


Self-organised

Title Date Place
Apicowplexa 2013 31.10.2013 Kusadasi, Turkey

Awards

Title Year
Best oral communication, Apicowplexa meeting, Edinburgh, Scotland 2015

Associated projects

Number Title Start Funding scheme
165782 The cross-talk between chemotherapy and immunity in murine and ovine neosporosis disease 01.04.2016 Project funding (Div. I-III)
184662 Effects of a double-edged sword: exploiting the interaction between immunity and chemotherapy in murine and ovine models of congenital neosporosis and toxoplasmosis 01.04.2019 Project funding (Div. I-III)
127374 Vaccines against neosporosis - studies in the mouse model 01.04.2010 Project funding (Div. I-III)

Abstract

Background: The apicomplexan Neospora caninum represents one of the major infectious causes of bovine abortion worldwide, and also causes fetal loss in sheep and other ruminants. Tachyzoites represent the proliferative stage and invade a large number of host cell types and tissues, leading to disease. Proteins associated with, and secreted from, specialized organelles such as rhoptries, micronemes and dense granules play a major role in the host invasion, and represent highly interesting targets for intervention. The goal of this project is to develop vaccines and drugs that prevent N. caninum infection and eliminate fetal loss due to neosporosis. These findings could be potentially also applied to other closely related apicomplexans such as Toxoplasma gondii and Besnoitia besnoiti. Working hypothesis: We propose that targeting the host cell invasion process by vaccination and chemotherapeutical means will lead to a significant reduction in parasite load in infected animals. This will be exploited for the development of novel preventive and therapeutic strategies. Specific aims: (1.) Prevention of experimental infection in the pregnant mouse model by vaccination employing recombinant rhoptry and microneme antigens expressed in a novel OprI-lipoprotein expression system, which allows for optimization of antigen presentation and thus improvement of vaccine efficacy.(2.) Development of a chemotherapeutical treatment strategy in N. caninum infected mice based on inhibitors of host cell invasion, such as calcium dependent kinase (CDPK) inhibitors and arylimidamides (pentamidine-derivatives).(3.) Development of an experimental N. caninum infection model in sheep to mimic the situation in cattle more accurately, and application of this model in vaccination and chemotherapy. Experimental design and/or methods: (1.) Vaccination: in order to improve antigen presentation and stimulation of the immune response, promising recombinant antigens (NcMIC1, NcMIC3, NcROP2) will be cloned and expressed in a novel expression system based on the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane. Respective recombinant fusion proteins will be investigated for protective activity against N. caninum challenge infection in BALB/c mouse models, and accompanying immunological parameters will be evaluated.(2.) Chemotherapy: novel inhibitors of calcium dependent protein kinase 1 (NcCDPK1) specifically target host cell invasion, and some arylimidamides also inhibit host cell entry in vitro. Newly synthesized CDPK inhibitors will be assessed in vitro, and the lead compound 1294 will be characterized with respect to its in vivo activity in both the non-pregnant and pregnant mouse model for N. caninum infection, either alone or in combination with selected arylimidamides. (3.) N. caninum infection model in sheep: pregnant sheep will be infected with N. caninum tachyzoites. Baseline parameters of the model in dams (clinical signs such as fever and abortion) and offspring (foetal death, stillbirth, birth of weak baby lambs, neurological disorders, congenital transmission in healthy lambs) will be standardized. Tissue samples of aborted offspring and/or newborn lambs will be analyzed by routine histological techniques, nested-PCR, real time PCR and immunohistochemistry (IHC) for measuring parasite and lesion presence and infection intensity. Other analyses include cellular and humoral peripheral responses and comparative detection of cytokines in placental tissues by real time PCR and IHC. Once this model is established, these parameters are assessed in pregnant and infected sheep vaccinated with the combination vaccine (recNcMIC1+NcMIC3+NcROP2) that showed promising results in the mouse model. Prospectively, this sheep model will be further developed to study endogenous transplacental infection and effects of interesting chemotherapeutic agents. Expected value of the project: This project has a high socio-economic value. By targeting the host cell invasion process of the invasive stage of the parasite, it aims to develop novel tools (selected vaccines and drugs) to limit fetal infection and abortion due to neosporosis. The mouse model will provide proof-of-concept, but not definitive results, since it does not accurately reflect the situation in cattle. We will overcome this bias by performing corresponding experiments in sheep as a more realistic alternative, albeit less expensive than cattle. This will lay the basis for a possible future application of vaccines and drugs in cattle. Thanks to the identification of novel vaccines and drugs in these parasites we will also gain further insight into the complex biology of intracellular parasitism and the host-parasite relationship. Thus, the expected output will prospectively lead to a significant reduction in economical losses due to neosporosis, and will be of high interest also for other apicomplexan disease models such as toxoplasmosis and besnoitiosis.
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