Browsing by Subject "Infektion"

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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.
Interaktion des Portalring-Proteins gp20 des Bakteriophagen T4 mit Wirtsproteinen von Escherichia coli
(2012) Quinten, Tobias; Kuhn, Andreas
Bacteriophage T4 is composed of the three structural subunits i. e. capsid, tail and tail fibres. Because of its contractible tail T4 is a member of the Myoviridae. A characteristic feature of its morphogenesis is the membrane-associated assembly of the head structure during a wild-type infection of E. coli. The portal protein, gp20, and the phage-chaperone gp40 are used to form a membrane-bound complex. Most likely, also proteins from the phage-host E. coli are involved in this process. This complex is the starting point for the assembly of the head-related core and scaffold structure. The mature head detaches from the membrane and is filled with DNA thereafter. In the context of this doctoral thesis the role and function of the portal protein gp20 and its interactions with cellular proteins was analyzed. A His-tag was fused to gp20 and it was purified by nickel-affinity chromatography. Also, interactions with the cellular chaperones DnaK, GroEL, Tig and YidC and the phage-chaperone gp40 were detected after formaldehyde crosslinking. Further studies of the cellular localization showed that gp20 and the fusion protein gp20-GFP are membrane-bound. The importance of YidC and DnaK for this membrane-association was demonstrated by fluorescence microscopy. Phage propagation was not affected by YidC depletion, whereas the loss of DnaK led to a reduced propagation. Prohead-structures, that are an intermediate stage of the capsid assembly, were isolated in YidC-free E. coli-cells membrane-unbound when infected with a phage mutant. Previous studies had led to the isolation of different amber mutants. Mutant amber20E481 was used in this thesis to analyze the assembly process in more detail. Here, under non-permissive conditions a 14 amino acid shortened protein, gp20s, is synthesized. Despite the fact that the capsid assembly is blocked in the non-suppressor strain, the localization and expression of the truncated protein was comparable to the wild type gp20. Overexpressed and His-tagged gp20s was found to crosslink with YidC, GroEL and gp40. Structural studies with a transmission electron microscope showed, that mature proheads were found and these were not filled with phage DNA. Most probably, a malfunction of gp20s during DNA packaging accounts for this.
Microbiological and proteome analysis to gain insights into the pathogenesis of the highly adapted not-cultivated hemotrophic Mycoplasma suis
(2016) Dietz, Stefanie; Hölzle, Ludwig
The aims of this work were to establish a chronic pig infection model and to clarify unrecognized transmission pathways of M. suis. In addition, proteomics-based investigations of M. suis should be performed to improve the knowledge on the host pathogen interactions and host adaptation in IAP. Based on the succeeded and frequently applied splenectomized M. suis pig model (acute infection model) it was possible to establish a novel non-splenectomized M. suis pig model. This infection model enables the experimental investigation of the chronic M. suis infection. To this end, the piglets were infected intramuscularly with a highly virulent M. suis strain. Infected animals exhibited clinical signs (e.g. Morbus maculosus) including the typical cyclic course of chronic IAP. In the next step, potential transmission pathways of M. suis were analyzed during acute and chronic M. suis infection based on these two pig infection models. Feces, urine, air and dust as well as nasal, vaginal and saliva excretions were collected during the course of infection and examined for M. suis DNA by Rt-PCR. For the first time it was possible to detect M. suis in urine with and without erythrocytes as well as in nasal, vaginal and saliva excretions during acute and chronic infections. These results indicate blood-independent M. suis transmission via vaginal discharge, nasal excretions, saliva, and urine. The non-culturability limited the improvement of proteomic-based investigations of M. suis-related host-pathogen interactions. Therefore, we used modern and sophisticated proteome analysis to solve this problem. Blood samples from experimentally infected pigs at different time points of infection were investigated. For this, novel enrichment methods for M. suis proteins (especially membrane proteins) were established. These methods enabled an improved resolution of the protein expression profile of M. suis and thereby deeper insights into the pathogenesis of this microorganism. Despite of the missing cultivation system it was possible to identify more than 50% of the predicted M. suis proteins during acute infection. This identification ratio is similar to the one found in cultivable bacteria. Furthermore, the results of the proteome analysis indicate that nutrients such as glucose, hexose-6-phosphate, spermidine, putrescine, phosphate, amino acids, magnesium, potassium, sodium and iron are taken up by M. suis from the host leading to the high degree of host adaptation. Therefore, gained information on expressed M. suis proteins involved in transport are helpful in the establishment of an in vitro cultivation system in future. Particularly the supplementation of individual nutrients can play key functions in the media to support growth. Besides the M. suis proteome the acquired dataset firstly enables also the quantitative identification of Sus scrofa proteins differentially expressed during M. suis infection. This information can be used to unravel infection-relevant processes in the host in further studies.