Browsing by Subject "Salmonella typhimurium"

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Vakzinierungsstrategien gegen eine Echinococcus multilocularis-Infektion zur Charakterisierung protektiver Immunantworten
(2010) Wassermann, Torsten; Mackenstedt, Ute
The larval stages of Echinococcus multilocularis are the causative agents of the human alveolar echinococcosis (AE), which is according to the WHO the most important parasite-induced zoonosis in middle Europe. Additional knowledge about the infection process and possible protective mechanisms against an infection with this cestode would be of great value. Almost all previous studies about Echinococcus multilocularis infections in the intermediate host were carried out by using the secondary echinococcosis as route of infection. This route of infection doesn?t correspond to the natural route, the oral uptake of Echinococcus eggs resulting in a primary alveolar echinococcosis. Thereby the resulting immune responses of the secondary AE cannot be converted without reservations to the primary AE, especially in the early stages of the infection. On that account the primary AE was carried out in this study. On the one hand a conservative vaccination with purified antigen EM95 and EMGAPDH in combination with an adjuvant was subcutaneous administered and on the other hand a system was developed in which the antigen EM95 were expressed by salmonellae and exported via the hemolysinA-transport system. A significant reduction of the formation of cysts could be achieved in two separate immunisation trials with EMGAPDH combined with the adjuvant Saponin which was administered subcutaneously. The manifestation of cysts was reduced by 76.4 % in the first and 86.1 % in the second trial compared to the infected control group. The subcutaneous immunisation with the antigen EM95 reduced the manifestation of cysts even by 96.9 % and 98.4 % respectively. Sixty percentages of the animals showed a complete protection against an infection with E. multilocularis in both trials. Based of the experiments which were carried out during this study, the influence of the secretory unit or the transmembrane domain of EM95 can be excluded. The subcutaneous immunisation with the recombinant antigens EM95 and EMGAPDH induced a high antibody titre against those proteins, which based predominantly on the IgG-subclasses IgG1 and IgG2a. The detection of specific antibodies against E. multilocularis crude antigen after duration of infection of 4 weeks was neither in protected animals nor in the infected control group possible. The vaccination trials with the recombinant antigens EM95 and EMGAPDH lead us to the conclusion that the murine immune system was stimulated to detect suitable target epitopes of the oncosphere surface and to destroy these oncospheres by classical complement activation. It seems very probable that cytotoxic T-cells and natural killer cells are involved in the protective immune mechanisms against an E. multilocularis infection owing to the finding of an increased release of IFNγ as a result of an immunisation with the recombinant antigens EM95 and EMGAPDH. Summarizing the investigated immunological parameters, the protective mechanisms are based on a Th1 associated immune response. Nevertheless, an involvement of Th2 components is conceivable due to the fact that at least in parts of the study the cytokine IL-10 and a distinct IgG1 antibody response could be detected past the immunisation. By the use of the plasmid pVDL9.3 we succeeded for the first time to manipulate Salmonella typhimurium to export the metazoan protein EM95 via the hemolysinA-system. The immunisation with ZpVDL9.3EM95 resulted in a reduction of manifested cysts by 78 % compared to the infected control group. Mice immunized with ZpVDL9.3EMGAPDH, where an export was not accomplished, showed a decrease in cysts manifestation by 73 %. The amount of cysts could be further reduced up to 87 % by the combined immunisation with the Salmonella vector ZpVDL9.3EMGAPDH and a subcutaneous application of EMGAPDH. A significant reduction of cysts (68 %) could be also observed by the control immunisation with plain S. typhimurium. Antibodies against the E. multilocularis antigens EM95 and EMGAPDH could not be detected after the immunisation with the Salmonella vectors. However, the antibody formation against S. typhimurium was strongly developed. A subcutaneous post infection immunisation trial with EM95 in combination with the adjuvant Saponin at the time of 4dpi, 7dpi, 24dpi and 60dpi led neither to a reduction of the amount of cysts nor to a change in size of the developed cysts. The gained insights of this study into the protective potential of EM95 and EMGAPDH on the basis of a natural infection rout expand the knowledge and understanding about the protective immune mechanisms against an Echinococcus multilocularis infection. The development of an immunisation system in form of a Salmonella typhimurium live vaccine, with the ability of exporting metazoan antigens, opens up new possibilities of immunisation strategies against diverse parasites in future.