Bockl K.,University of Regensburg |
Wild J.,University of Regensburg |
Bredl S.,University of Regensburg |
Bredl S.,Redbiotec |
And 5 more authors.
HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ + CD8 + T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2 d T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials. © 2012 Böckl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source
Eberhard R.,University of Zurich |
Stergiou L.,University of Zurich |
Stergiou L.,Redbiotec |
Hofmann E.R.,University of Zurich |
And 8 more authors.
Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. © 2013 Eberhard et al. Source
Sendoel A.,University of Zurich |
Sendoel A.,Rockefeller University |
Sendoel A.,Redbiotec |
Maida S.,German Center for Neurodegenerative Diseases |
And 14 more authors.
Nature Cell Biology
Microtubule-targeting chemotherapeutics induce apoptosis in cancer cells by promoting the phosphorylation and degradation of the anti-apoptotic BCL-2 family member MCL1. The signalling cascade linking microtubule disruption to MCL1 degradation remains however to be defined. Here, we establish an in vivo screening strategy in Caenorhabditis elegans to uncover genes involved in chemotherapy-induced apoptosis. Using an RNAi-based screen, we identify three genes required for vincristine-induced apoptosis. We show that the DEP domain protein LET-99 acts upstream of the heterotrimeric G protein alpha subunit GPA-11 to control activation of the stress kinase JNK-1. The human homologue of LET-99, DEPDC1, similarly regulates vincristine-induced cell death by promoting JNK-dependent degradation of the BCL-2 family protein MCL1. Collectively, these data uncover an evolutionarily conserved mediator of anti-tubulin drug-induced apoptosis and suggest that DEPDC1 levels could be an additional determinant for therapy response upstream of MCL1. © 2014 Macmillan Publishers Limited. All rights reserved. Source
Ries C.,ZHAW Zurich University of Applied Sciences |
John G.,PreSens Precision Sensing GmbH |
John C.,Redbiotec |
Eibl R.,ZHAW Zurich University of Applied Sciences |
Eibl D.,ZHAW Zurich University of Applied Sciences
Engineering in Life Sciences
While wave-mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture-based production at liter-scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter-scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k La-values) for PreSens 250mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation-free growth of insect cells up to high cell densities in batch mode (1.6×10 7 cells/mL) and for the efficient expression of an intracellular model protein. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Source
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2013.2.3.0-1 | Award Amount: 6.09M | Year: 2013
As current influenza vaccines only afford limited protection against seasonal as well as pandemic influenza, and require regular updating, the development of a universal influenza vaccine that can provide broad coverage against different strains within a subtype or even across subtypes has become a key health care priority in both industrialized and low and middle income countries. EDUFLUVAC aims to develop an influenza vaccine that educates the immune system to recognise common influenza epitopes, using a combination of influenza haemagglutinin (HA) antigens delivered on a single virus-like particle. This vaccine concept, using the proven, modern technology of baculovirus VLPs will enable enhanced cross linking of B-cell receptors and favour induction of B-cell responses against common epitopes essential for long-lasting cross recognition and protection. The mechanism underlying the broadening of antibody responses is the increased relative concentration of common epitopes diluting out strain specific epitopes. This will be achieved by testing the ability of a combination of historic HA variants to protect against a variety of modern isolates. A similar approach for developing a novel vaccine for pandemic use will be tested. The knowledge generated will provide Proof of Principle in relevant animal models for an influenza vaccine that does not have to be updated annually, does not have to be manufactured on an annual basis and will not require an annual vaccination campaign. It will also provide for an economically viable large scale production platform for influenza vaccine which, in the event of a pandemic, can manufacture rapidly high quantities of vaccine. All industrial partners (including two SMEs) will benefit from the compilation of the regulatory dossier for transfer to larger vaccine companies and the deeper knowledge integration emanating from EDUFLUVAC in a $2.8 billion global annual influenza vaccine market.