Abstract
(Englisch)
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Tropical theileriosis, a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria annulata, is an economically important disease which threatens an estimated 250 million cattle in regions of North Africa, Southern Europe and Asia. Current control measures against tropical theileriosis Include vaccination with attenuated macroschizont-infected cell lines, the infection and treatment method or acaricides against the vector. All these methods have significant drawbacks, particularly under the tropical conditions in which they are applied;for exemple the possibility of attenuated parasites reverting to need of a cold chain delivery system, and the coot of chemotherapy. Due to this, current research is aimed at synthesising a recombinant subunit vaccine that will alleviate these problems. It is now generally accepted that the protective immunity in the case of T. annulata infections is based on a cellular T cell response. One major problem is the identification of antigens, stimulating these protective cellular responses. Secreted parasite antigens are important vaccine candidates as they are subjected to processing and MHC presentation by the infected cell.
One potentially interesting antigen ist the T. annulata antigen 11 E, which we have identivied recently. A Genbank homology search with previously identified T. parva 11 E revealed significant similarity to members of the thioredoxin family. Highest homology was found around the active centre of this enzyme. In order to further characterise 11E and to determine ist function, we attempted to inhibit its function in infected cells by a dominant-negative approach. Site directed mutagenesis was used destroy the active centre of recombinant T. pava 11 E. A plasmid coding for the mutated 11 E was transfected and expressed in T. annulata-infected cells. We then tested the effects by a viability assy. First results indicate that cells undergoprogrammed cell death (apoptpsis) when they overexpress the mutated 11 E. In order to localise mutated and wildtype 11E in transfected cells, we used non-parasitised Cos cells for transfection experiments. Unexpectedly, the mutated 11E was found to colocalise with the mitochondria of the transfected cell, whereas wildtype 11E was found predominantly in the nucleus and the cytoplasm of the transfected cells. An explanation, for this observation could be that the wildtype 11E with an Intact active centre is retained in the cytoplasm by interaction with other proteins and is subsequently into the nucleus. The mutated 11E, however, cannot bind other proteins because the active has been destroyed. Since it still contains a mitochondrial targetting sequence it will be directed to this cellorganelle. The mitochondrial localisation does not reflect the situation in parasitised cells. Therefore, we will try to modify the amino acids of the recombinant 11E, which are responsible for the mitochondrial targeting and test the new constructs again in transfection experiments.
By Immunoflourescence studies we have identified another parasite molecule of T. annulata which could be highly interesting, A commercially available monoclonal antibody directed against the HA-tag stained the intracellular T. annulata schizont and the host cell cytoplasm intensively. If we can confirm that the protein found in the cytoplasm is of parasite origin, this molecule could be another interesting subunit vaccine candidate. As a specific monoclonal antibody is already available, it should be feasible to evaluate the corresponding molecule in a short time. We will pass the information and the necessary reagents to our EU partners if this protein is indeed a secreted parasite molecule.
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