Developing novel strategies for oncolytic adenovirus therapy by host cell gene expression profiling and arming with therapeutic antibodies

Die virale Onkolyse oder Virotherapie ist ein vielversprechender Ansatz zur Behandlung von Krebserkrankungen und wurde unter anderem klinisch getestet für onkolytische Adenoviren. Jedoch schwankt die therapeutische Wirksamkeit von Fall zu Fall, was daran liegen könnte, dass Krebszellen nicht die natürlichen Wirtszellen von Adenoviren sind. Ziel meiner Doktorarbeit war es Adenovirus 5 Infektionen in ihrem natürlichen Umfeld primärer Bronchialepithelzellen und verschiedener Krebszellen zu untersuchen, um Gene und zelluläre Signalwege zu finden, die einen Einfluss auf die adenovirale Replikation haben. Unter anderem konnte ich zeigen, dass in Bronchialepithelzellen die frühe virale Genexpression und DNA Replikation sehr schnell einsetzt, verbunden mit einer hohen Zytotoxizität. Dagegen war die frühe virale Genexpression als auch die DNA Replikation in zwei Melanomzelllinien stark verlangsamt, verbunden mit einer geringen Zytotoxizität. Genexpressionsanalysen mittels Microarray in infizierten Bronchialepithelzellen und jener Melanomzelllinien konnten zeigen, dass in den jeweiligen Zelltypen unterschiedliche Expressionsmuster einzelner Gene und zellulärer Signalwege vorlagen. Der am stärksten beeinflusste Signalweg reguliert den Eintritt in die S-Phase des Zellzyklus und war in den primären Bronchialepithelzellen aktiviert, aber nicht in den Melanomzelllinien. Anhand der Daten konnte eine Strategie zur verbesserten Onkolyse in einzelnen Melanomzelllinien entwickelt werden. Dafür wurden Adenovirus infizierte Melanomzellen zusätzlich mit dem Chemotherapeutikum Temozolomid behandelt. Ein weiteres Ziel meiner Arbeit war es, die Wirksamkeit onkolytischer Adenoviren durch die Expression von therapeutischen Antikörpern zu verbessern. Dafür habe ich tumorspezifische Adenoviren mit einem rekombinanten „single-chain“ Antikörper-Gen ausgerüstet. Die Antikörper erkennen das Tumor-assoziierte karzinoembryonale Antigen und verfügen außerdem über die Effektordomäne des Typ G Immunglobulins 2a, um eine Antikörper vermittelte Immunantwort gegen Krebszellen zu richten. Zuerst wurden verschiedene Expressionskonstrukte für eine optimale Antikörperproduktion mit dem adenoviralen „major late promoter“ getestet. Dabei wurden zwei effiziente Expressionskonstrukte identifziert, die sogenannte „internal ribosomal entry site“ und ein Spleiß-Akzeptor des humanen Adenovirus 40. Anschließend wurden die rekombinant hergestellten Antikörper im Western Blot, ELISA und mittels Durchflusszytometrie auf ihre Funktionalität geprüft. Eine erfolgreiche Antikörper-Antigen Reaktion konnte in-vitro als auch anhand lebender Tumorzellen gezeigt werden

Energy-scaling of the product state distribution for three-body recombination of ultracold atoms

Three-body recombination is a chemical reaction where the collision of three atoms leads to the formation of a diatomic molecule. In the ultracold regime it is expected that the production rate of a molecule generally decreases with its binding energy $E_b$, however, its precise dependence and the physics governing it have been left unclear so far. Here, we present a comprehensive experimental and theoretical study of the energy dependency for three-body recombination of ultracold Rb. For this, we determine production rates for molecules in a state-to-state resolved manner, with the binding energies $E_b$ ranging from 0.02 to 77 GHz$\times h$. We find that the formation rate approximately scales as $E_b^{-\alpha}$, where $\alpha$ is in the vicinity of 1. The formation rate typically varies only within a factor of two for different rotational angular momenta of the molecular product, apart from a possible centrifugal barrier suppression for low binding energies. In addition to numerical three-body calculations we present a perturbative model which reveals the physical origin of the energy scaling of the formation rate. Furthermore, we show that the scaling law potentially holds universally for a broad range of interaction potentials.Comment: 15 pages, 13 figure

Replication and Virus-Induced Transcriptome of HAdV-5 in Normal Host Cells versus Cancer Cells - Differences of Relevance for Adenoviral Oncolysis

Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication

Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors▿

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures

Extracellular forms of Mycobacterium bovis BCG in the mucosal lymphatic tissues following oral vaccination

Oral vaccination with BCG provides protective systemic immunity against pathogenic mycobacterial challenge. In this study, the anatomical distribution of Mycobacterium bovis BCG following oral vaccination was investigated. Replicating bacteria in the Peyer's patches and mesenteric lymph nodes were present as solitary rods or clusters of two to three bacteria, the majority of which were isolated ex vivo as extracellular forms. Only a minority were shown to be associated with typical antigen-presenting cells. Acid-fast staining of mast cell granules in lymphoid tissues revealed a potential pitfall for these analyses and may explain previous reports of acid-fast ‘coccoid’ forms of mycobacteria in tissues