Publicaties

Identification of novel Theileria parva candidate vaccine antigens and discovery of new therapeutic drugs for the control of East Coast fever

Nyagwange, James J.

Samenvatting

ECF caused by the hemoprotozoan pathogen Theileria parva is the leading tick-borne disease of cattle in sub-Saharan Africa, causing death of cattle and significant economic losses. Currently control of the disease is dependent on vaccination and drug treatment. The infection and treatment method of vaccination involves deliberate infection with live sporozoites from three strains namely T. parva Muguga, Kiambu5 and Serengeti (known as Muguga cocktail) and simultaneous treatment with a long-acting oxytetracycline. Although effective, this method has serious limitations; the immunity generated by the vaccine is strain specific and immunized cattle can become life-long asymptomatic carriers of the parasite, posing risk for the spread of the disease through introduction of ‘foreign’ T. parva strains in areas where the strains in the vaccine are not present. The production and distribution of the vaccine involves complex procedures requiring infrastructure and specialized expertise which are not always available. These concerns undermine the widespread uptake of this vaccine. There have been efforts to identify sporozoite antigens that are targets of neutralizing antibodies and one protein, p67 is considered the lead candidate for development of a subunit vaccine. However, this protein only protects only a proportion of immunized cattle. Although research is ongoing on different formulations and immunization regimen to improve the efficacy of this antigen, protection against ECF might be improved by additional sporozoite antigens that induce sporozoite neutralizing antibodies. In an attempt to identify such antigens and to support research on the biology of T. parva we employed two strategies. Firstly, we characterized the sporozoite proteome by LC-MS/MS analysis. In total, 4,780 proteins were identified in an enriched preparation of sporozoites. Of these, 2,007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2,773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to ECF. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Secondly, we searched the re-annotated T. parva genome for genes predicted to contain GPI anchor signals, since they are likely to be located on the cell surface and expressed fragments of six of the selected genes in E. coli. The recombinant proteins were used to raise antisera in mice. Antisera to two proteins, TpMuguga_01g00876 and TpMuguga_01g00939, neutralized sporozoite infectivity to a high degree, while antisera to two additional proteins, TpMuguga_01g00095 and TpMuguga_04g00437, exhibited moderate neutralizing capacity. We conclude that these four antigens are potential vaccine candidates, which should be evaluated further in cattle.

Chemotherapy is another method of ECF control and is currently dependent on the use of buparvaquone, a drug that was developed in the late 1980’s and to which resistance has been reported in the related parasite Theileria annulata. Fortunately, resistance has not been reported in T. parva, but this could occur, underscoring the urgency for discovery of new cost-effective drugs. Using a 3H-thymidine incorporation assay we screened the Medicines for Malaria Venture (MMV) open source 400 malaria box and 396 pathogen box compounds to discover novel compounds with anti-parasitic activity. We identified nine malaria box compounds and eight pathogen box compounds that inhibit the proliferation of a T. parva infected cell line. However, only two compounds, MMV008212 (malaria box) and MMV688372 (pathogen box) represent promising leads with half maximal inhibitory concentration (IC50) values of 0.78 and 0.61 µM, respectively, and half maximal cytotoxic concentration (CC50) values > 5 µM. The remaining selected compounds exhibited a high degree of non-specific cytotoxicity (CC50 values < 1.09 µM) on the proliferation of bovine peripheral blood mononuclear cells stimulated with concanavalin A. We also tested the anti-cancer drug, dasatinib, used in the therapy of some leukemias. Dasatinib was as active and safe as buparvaquone in vitro, with an IC50 of 5 and 4.2 nM, respectively, and CC50 > 10 µM. Our preliminary data suggest that it may be possible to repurpose compounds from the cancer field as well as MMV malaria and pathogen boxes.