Entwicklung neutralisierender und nicht-neutralisierender Antikörper gegen bekannte und neue Epitope auf Clostridioides difficile Toxin B

In this study, antibody phage display technology was used to generate a broad panel of fully human monoclonal antibodies that target TcdB, one of the main virulence factors of Clostridioides difficile and target of new therapeutic approaches. TcdB has a complex domain structure and intoxicates cells via a multistep mechanism, thus offering several weak points which can be addressed with antibodies. Applying different panning strategies against either full TcdB, truncated variants or single domains, 36 unique scFvs were generated of which 31 were further characterized after conversion into the bivalent scFv-Fc format. ELISA and Immunoblot analysis proved TcdB binding and domain specificity of these 31 antibodies analyzed, and confirmed the selection of antibodies with diverse binding characteristics. Using a cell-based in vitro assay, all antibodies were screened for neutralization of TcdB induced cell rounding. Here two antibodies efficiently neutralized TcdB. To test whether in vitro neutralization could be further improved by targeting different domains of TcdB at the same time, antibody combinations were tested in in vitro neutralization of TcdB induced cell rounding. Unfortunately, for the combinations tested in this study, the improvement of neutralization observed was rather of additive than synergistic nature. To gain more information about the binding sites of the antibodies two independent approaches of epitope mapping were followed. With the combination of Peptide array and TcdB-fragment phage display, novel and already described epitopes on TcdB could be identified. By correlating these epitopes with the in vitro neutralization efficacy, a new epitope within the glucosyltransferase domain of TcdB was identified which conveys neutralization. Thus, this study provides valuable information for further development of therapeutics against TcdB. Also, the two potent neutralizing antibodies might be candidates for further preclinical development. Future therapeutic use of these antibodies, preferably as antibody combination with antibodies targeting different domains or epitopes, is easily conceivable due to the human origin of these antibodies.In dieser Studie wurde die Antikörper-Phagen-Display-Technologie zur Generierung vollständig humaner monoklonaler Antikörper gegen TcdB, einen der wichtigsten Virulenzfaktoren von Clostridioides difficile, eingesetzt. TcdB hat eine komplexe Domänenstruktur und vergiftet Zellen über einen mehrschrittigen Mechanismus. Somit umfasst es mehrere Regionen, an denen Antikörper angreifen können um das Toxin zu neutralisieren. In mehreren Bio-Pannings, entweder gegen das vollständige TcdB, verkürzte Varianten oder einzelne Domänen, wurden 36 einzigartige scFvs generiert, von denen 31 nach Konvertierung in das bivalente scFv-Fc-Format weiter charakterisiert wurden. Mittels Antigen-ELISA und Immunoblot konnte die TcdB-Bindung dieser 31 Antikörper validiert und die Domänenspezifität bestimmt werden. Über einen zellbasierten in vitro Assay wurden die Antikörper auf Neutralisierung der durch TcdB-induzierten Zellrundung untersucht. Zwei der Antikörper neutralisieren TcdB besonders effizient. Durch die simultane Adressierung verschiedener Domänen von TcdB mit Antikörperkombinationen konnte die in vitro Neutralisierung der durch TcdB-induzierten Zellrundung weiter verbessert werden. Die beobachtete Verbesserung der Neutralisierung war allerdings eher auf additive als auf synergistische Effekte zurückzuführen. Des Weiteren wurden zwei unabhängige Ansätze zur Epitop Kartierung der Antikörper verfolgt. Mit der Kombination von Peptid-Array und TcdB-Fragment-Phagen-Display konnten neue sowie bereits beschriebene Epitope auf TcdB identifiziert werden. Durch Korrelation der Epitope mit der Neutralisierungseffizienz der gegen sie gerichteten Antikörper, konnte ein neues Epitop innerhalb der Glucosyltransferase-Domäne identifiziert werden welches Neutralisierung vermittelt. Somit liefert diese Studie wertvolle Informationen für die weitere Entwicklung von Therapeutika gegen TcdB. Auch die beiden potenten neutralisierenden Antikörper sind mögliche Kandidaten für eine weitere präklinische Entwicklung. Ein therapeutischer Einsatz dieser Antikörper, vorzugsweise als Antikörper-Kombination mit Antikörpern, die gegen andere Domänen oder Epitope gerichtet sind, ist aufgrund des menschlichen Ursprungs dieser Antikörper gut vorstellbar

Incorporating linkage learning into the GeLog framework

This article introduces modifications that have been applied to GeLog, a genetic logic programming framework, in order to improve its performance. The main emphasis of this work is the structure processing of genetic algorithms. As studies have shown, the linkage of genes plays an important role in the performance of genetic algorithms. Thus, different approaches that take linkage learning into account have been reviewed and the most promising has been implemented and tested with GeLog. It is demonstrated that the modified program solves problems that proved hard for the original system

Observation of Fano-Resonances in Single-Wall Carbon Nanotubes

We have explored the low-temperature linear and non-linear electrical conductance $G$ of metallic carbon nanotubes (CNTs), which were grown by the chemical-vapor deposition method. The high transparency of the contacts allows to study these two-terminal devices in the high conductance regime. We observe the expected four-fold shell pattern together with Kondo physics at intermediate transparency {G\alt 2e^2/h} and a transition to the open regime in which the maximum conductance is doubled and bound by $G_{max}=4e^2/h$. In the high-$G$ regime, at the transition from a quantum dot to a weak link, the CNT levels are strongly broadened. Nonetheless, sharp resonances appear superimposed on the background which varies slowly with gate voltage. The resonances are identified by their lineshape as Fano resonances. The origin of Fano resonances is discussed along the modelling.Comment: pdf including figures, see: http://www.unibas.ch/phys-meso/Research/Papers/2004/Fano-CVD-SWNT.pd

Magneto-transport investigations on multi-electron quantum dots : Coulomb blockade, Kondo effect, and Fano regime

[no abstract

Charge dynamics effects in conductance through a large semi-open quantum dot

Fano lineshapes in resonant transmission in a quantum dot imply interference between localized and extended states. The influence of the charge accumulated at the localized levels, which screens the external gate voltage acting on the conduction channel is investigated. The modified Fano q parameter and the resonant conduction is derived starting from a microscopic Hamiltonian. The latest experiments on "charge sensing" and ``Coulomb modified Fano sensing `` compare well with the results of the present model.Comment: 5 pages, 4 figures, RevTex styl

Pulse Propagation in Resonant Tunneling

We consider the analytically solvable model of a Gaussian pulse tunneling through a transmission resonance with a Breit-Wigner characteristic. The solution allows for the identification of two opposite pulse propagation regimes: if the resonance is broad compared to the energetic width of the incident Gaussian pulse a weakly deformed and slightly delayed transmitted Gaussian pulse is found. In the opposite limit of a narrow resonance the dying out of the transmitted pulse is dominated by the slow exponential decay characteristic of a quasi-bound state with a long life time (decaying state). We discuss the limitation of the achievable pulse transfer rate resulting from the slow decay. Finally, it is demonstrated that for narrow resonances a small second component is superimposed to the exponential decay which leads to characteristic interference oscillations.Comment: 6 pages, 4 figure

Flux-quantum-modulated Kondo conductance in a multielectron quantum dot

We investigate a lateral semiconductor quantum dot with a large number of electrons in the limit of strong coupling to the leads. A Kondo effect is observed and can be tuned in a perpendicular magnetic field. This Kondo effect does not exhibit Zeeman splitting. It shows a modulation with the periodicity of one flux quantum per dot area at low temperatures. The modulation leads to a novel, strikingly regular stripe pattern for a wide range in magnetic field and number of electrons.Comment: 4 pages, 5 figure

The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding

Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin–receptor interaction