Neospora caninum; recombinant antigens; rhoptries; calcium dependent protein kinase inhibitors; invasion; chemotherapy; micronemes; vaccine; Apicomplexa
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Hemphill Andrew, Leitão Alexandre (2014), ApiCOWplexa 2013--2nd International Meeting on Apicomplexan Parasites in Farm Animals., in Parasitology
, 141(11), 1355-8.
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.
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.
Monney Thierry, Hemphill Andrew (2014), Vaccines against neosporosis: what can we learn from the past studies?, in Experimental parasitology
, 140, 52-70.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.