Browsing by Subject "Bacillus anthracis"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Publication
    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.
  • Thumbnail Image
    Publication
    Herstellung monoklonaler Antikörper gegen thermostabile Antigene von Bacillus anthracis zur Anwendung in der Anthraxdiagnostik
    (2012) Hilss, Karen; Beyer, Wolfgang
    The Ascoli test is a fast and inexpensive diagnostic tool, using polyclonal serum against thermostable antigens of B. anthracis. However, this test is not highly specific for B. anthracis, since the thermostable antigens, on which this test is based, are also present in other Bacillus species and therefore lead to cross-reactivity. By employing monoclonal antibodies against B. anthracis specific thermostable antigens, the cross-reactivity with other Bacillus species could be eliminated. The aim of this study was to generate monoclonal antibodies, which react specifically with the potential B. anthracis specific thermostable antigens. At the beginning thermostable antigen preparations from vegetative B. cereus and B. anthracis cultures, as well as from B. cereus and B. anthracis spores were prepared. These eight antigen preparations were used to immunise rabbits. The resulting polyclonal antisera were used to determine cross-reactivity between B. cereus and B. anthracis in Western Blot analysis. In these cross-reactivity tests two proteins with a molecular weight of approximately 30 and 50 kDa respectively, which are specifically present in antigen preparations of B. anthracis were identified. These proteins do not react with sera of rabbits, immunised with the B. cereus preparations. The 30 kDa protein is present in vegetative and spore preparations of B. anthracis, while the 50 kDa protein is only present in vegetative antigen preparations of B. anthracis. These potentially B. anthracis specific proteins in the vegetative antigen preparation of B. anthracis were partially purified with anion exchange chromatography using FPLC and were used to immunise three BALB/c mice. The spleen of the mouse with the highest specific antibody response was then used to fuse the B-cells with murine myeloma cells in order to generate hybridomas. The supernatants of the resulting hybridomas were screened to identify clones producing antibodies against the thermostable antigens of B. anthracis. After 14 screens the positive clones were divided into two different cell lines. The clones of the V- (vegetative) line were further tested for production of antibodies against the thermostable antigens of vegetative cells of B. anthracis. The S- (spores) line was screened for clones producing antibodies against B. anthracis spore preparations. After two and four screens, the three monoclonal cell lines BaV5, BaV15 and BaV16 were established. The determination of the immunoglobulin class revealed, that BaV5 is a mixed culture with several different antibodies. The cell lines BaV15 and BaV16 produce an immunoglobulin of class M. In determining the specificity of the monoclonal antibodies BaV15 and BaV16 purified from Squarix Biotechnology, no cross-reactivity with 20 vegetative and 10 spore preparations of different Bacillus ssp. (non-anthracis) was found. The purified antibodies, which specifically detect vegetative cells of B. anthracis, were found to be unstable. Trying to stabilize the antibody by additives led to no success, so that further analyses for characterization of the antibodies were not possible.