Browsing by Subject "Impfstoff"

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Entwicklung und Testung neuer DNA- und Protein-basierter Multikomponentenvakzinen sowie regulatorischer Adjuvanzien gegen eine Infektion mit B. anthracis in Auszucht-Mäusen und Ziegen
(2015) Köhler, Susanne Melanie; Beyer, Wolfgang
The discovery of the Sterne spore live vaccine (SSLV) and subsequently its application in a veterinary context contributed to the global reduction of Anthrax related outbreaks since 1930. Nonetheless the causative agent Bacillus anthracis is still prevalent in some mediterranean countries, South and Central America, Africa and Central Asia, as well as the USA and Canada. Reasons for this are the persistence of the pathogen in the soil, as well as still undefined factors for an ongoing cycle of outbreak and spread of the disease and the limited applicability of the SSLV. This includes the necessity to revaccinate annually, the residual virulence in certain sensitive species (e. g. goats and llamas) and the incompatibility to treat and vaccinate simultaneously. To participate in the ongoing search for alternative vaccines this work was dedicated to evaluate protein- and DNA-based components as potential ingredients for a multi-component non-living vaccine formulation (NLV). For the protein-based NLV these included rPA83 as part of the Anthrax toxin, rBclA and Formalin inactivated spores (FIS) as spore specific antigens, a Capsule-Lipopeptide conjugate as part of the vegetative form of the pathogen and a Lipopeptide-adjuvant. The DNA-vaccines consisted of vector-backbones comprising signal sequences able to direct the integrated antigens (rPA83, PAD4LFD1 and BclAD1D3) to the MHCI, MHCII and the secretory pathway. A sperate vector encoding for a positive MHCII-regulator (CIITA) and a vector internal sequence for the Interferon-ß promotor stimulator (mIPS1) served as adjuvants for the DNA-vaccines. The majority of the groups showed detectable antibody titres against their respective antigens, with protein vaccines generally eliciting higher titres against rPA83 than the DNA-vaccines. Regarding rBclA equivalent high titres were measured for protein- and DNA-vaccines alike, which also corresponded to the anti-FIS titres for groups immunized with rBclA, FIS or both. The Capsule-Lipopeptide conjugate did not elicit high titres against the capsule, possibly due to an immune suppressing epitope. Survival rates ranged between 10 and 100 %, with full protection only achieved in a combination of all antigens including FIS. All DNA-vectors induced 30 – 50 % protectiveness when given alone. Notably DNA-vectors including BclAD1D3 elicited 50 % survival and sterile immunity. A combination of the most promising vectors encoding for toxin and spore specific antigens achieved 90 % protectiveness in mice. According to the results from the mice trials, the auspicious protein- and DNA-vaccine combinations were tested in goats in comparison to the SSLV in cooperation with our project partners in South Africa and Turkey. The efficacy of the SSLV was assessed in 3 groups which were challenged shortly after the first immunisation, one year after the first immunisation or after the revaccination. Apart from the comparison of immunogenicity and protectiveness between SSLV and NLV in goats, assessment of data concerning the titre development of SSLV-immunized goats during the course of a year as well as detailed diagnostic data during the infection (behavior, temperature, bacterial loads, correlations and minimal infective dose) were integral part of this study. Compared to one another the SSLV-immunized animals showed equal or higher antibody titres against the measured antigens, with FIS and rPA83 being the most immunogen antigens. Utilizing a higher dose (75 µg) the protein-based NLV protected equivalently to the SSLV (60 – 100 %) yielding 50 % protectiveness without FIS and 80 % if FIS was included. The DNA-vaccines showed little to no immunogenicity in goats, thus no challenge was performed on these animals. The humoral reaction against BclA was generally poor in goats, which has not been noted before and could be a basis for further improvements concerning the SSLV and NLV alike. The different immunizations with the SSLV revealed a broad range for the efficacy of the first vaccination as well as a notable difference in the antibody spectrum between first vaccination and revaccination. Together with the recorded data of the antibody titre development throughout a year a more optimal protocol for immunisation with the SSLV, possibly in combination with an NLV was postulated.
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.