Porcine endogene Retroviren und Xenotransplantation: Evaluierung des Infektionsrisikos und Strategien zur Hemmung der Virusreplikation mittels RNA-Interferenz

Der anhaltende Engpass an Allotransplantaten macht die Suche nach einer geeigneten Alternative dringend notwendig. Ein Lösungsansatz besteht in der Transplantation porciner Zellen oder Organe auf den Menschen. Derartige Xenotransplantationen bergen momentan jedoch noch etliche Risiken, wie immunologische Abstossungsreaktionen oder physiologische Unverträglichkeiten. Zudem besteht die Gefahr einer Übertragung von porcinen Mikroorganismen auf den humanen Rezipienten. Im Unterschied zu den meisten bakteriellen und viralen Erregern des Schweins, die durch Zucht unter keimfreien Bedingungen eliminiert werden können, ist das Genom der porcinen endogenen Retroviren mit zahlreichen Kopien stabil im Genom des Schweins integriert. Phylogenetisch verwandte Retroviren, wie die Leukämieviren der Maus (MuLV) und der Katze (FeLV) induzieren im infizierten Tier Leukämien, Tumore und Immundefizienzen und zeigen somit das Gefährdungspotential einer möglichen PERV-Infektion humaner Rezipienten. Zur Risikoevaluierung von Xenotransplantationen wurden daher im Rahmen dieser Arbeit in vivo Infektionsversuche durchgeführt. Bei den Untersuchungen mit Cynomolgusasen wurde vor allem darauf geachtet, zukünftige Xenotransplantationen beim Menschen weitestgehend zu simulieren: So wurden vergleichbare pharmakologische Immunsuppressiva appliziert, zusätzlich wurde das porcine Organ in direkter Nähe zu suszeptiblem humanem Gewebe transplantiert. Bei keinem der Tiere konnte eine PERV-Infektion des simianen oder humanen Gewebes festgestellt werden. Allerdings war die Ueberlebensdauer der Versuchstiere zu kurz, um verlässliche Aussagen über die Virustransmission machen zu können, da porcine Zellen, Gewebe und Organe bei künftigen Xenotransplantationen funktionell bis zu mehreren Jahren im Körper des Patienten verbleiben sollen. Auch bei einem zweiten in vivo Infektionsversuch, bei dem porcine Langerhanssche Inselzellen in Ratten mit chemisch induziertem Diabetes transplantiert worden waren, konnte keine PERV-Transmission nachgewiesen werden. Im Laufe weiterer Untersuchungen stellte sich allerding heraus, dass zumindest die analysierten Ratten-Fibroblasten unter in vitro Bedingungen nicht für porcine endogene Retroviren permissiv sind. Um Xenotransplantationen in Zukunft sicherer gestalten zu können, wurde erstmals das Potential des relativ neu entdeckten Phänomens der RNA-Interferenz hinsichtlich einer antiviralen Strategie gegen die Replikation von PERV analysiert: Anhand von neun unterschiedlichen synthetischen siRNAs gelang es, eine optimale Target-Sequenz in der pol-Region des PERV-Genoms (pol2) zu identifizieren, so dass die PERV Expression in PERV infizierten humanen 293-Zellen um mehr als 80% inhibiert werden konnte. Ausgehend von dieser pol2 siRNA-Sequenz wurde ein Plasmid mit einer shRNA-Expressionskassette konstruiert, das nach stabiler Transfektion eine starke und dauerhafte Hemmung der PERV-Expression hervorrief. Sowohl die mRNA, die viralen Proteine und vor allem die Menge freigesetzter infektiöser Viruspartikel war reduziert. Für die Herstellung transgener Schweine mit verminderter PERV-Expression empfiehlt sich der Einsatz lentiviraler Expressionsvektoren, um die shRNA-Expressionskassette effektiv in porcine Blastocysten transferieren zu können. Es gelang, ein entsprechendes auf HIV-1 basierendes lentivirales Vektorsystem herzustellen. Durch Infektion mit solchen nicht-replizierenden Viren konnte die Expression von PERV in humanen, aber auch in primären porcinen Zellen um 90% inhibiert werden. Somit konnte erstmals die Hemmung endogener Retroviren mittels RNA-Interferenz gezeigt werden. Mit diesen Ergebnissen ist der Grundstein für die Herstellung entsprechender transgener Tiere gelegt. Durch die Klonierung und Expression des p27Gag-Proteins und der Generierung spezifscher Antikörper gegen das rekombinante Gag-Protein sowie der Klonierung und Sequenzierung eines PERV-A/C Molekularklons wurden weitere Vorrausetzungen geschaffen, um das Risikopotential zukünftiger Xenotransplantationen hinsichtlich einer Virustransmission mit den porcinen endogenen Retroviren besser einschätzen zu können

A new porcine suspension cell line (PBG.PK-21) provides efficient production for influenza and yellow fever vaccine viruses

Shifting from egg-based influenza and yellow fever vaccine production towards efficient cell culture-based manufacturing is one of the biggest challenges in viral vaccine manufacturing. Innovative bioprocesses, such as high cell density [1] or continuous virus cultivations [2] improving cell-based viral vaccine production have been shown over the past years. However, finding the right high-yield cell substrate is still pending for many important viruses. Ideally, such host cell lines should enable the efficient production of several virus strains to set-up manufacturing platforms. Here, we report on a novel suspension cell line (PBG.PK-21 (ProBioGen) derived from immortal porcine kidney cells. These cells were first adapted to chemically defined virus production medium CD-U5 (ProBioGen) and agitated suspension culture. Despite its robust growth these cells were not yet suitable for virus manufacture due to a chronic infection with porcine circovirus 1 (PCV1) that is often found in porcine cell lines. The cells were cured from infection after siRNA mediated suppression of the contaminating virus followed by single cell cloning. Clone number 21 proven to be free from PCV1 over multiple passages and distinguished by high peak cell densities and good productivity for other viruses was chosen for further analysis. Cell growth, cell metabolism and virus production were characterized in shake flasks and bioreactors. Cell concentration up to 8 x 106 cells/mL and a doubling time of 33 h were obtained in batch mode in CD-U5 medium. Furthermore, process intensification using either semi-perfusion in shake flasks or hollow fiber-based perfusion with an ATF2 system coupled to a 0.6 L wv stirred tank bioreactor system was evaluated. In this system, cell densities up to 42 x 106 cells/mL were achieved with a cell-specific perfusion rate of 0.07 nL/cell/day. After optimization of influenza A/PR/8/34 (H1N1) virus production, HA titres of 3.3 log(HAU/100µL) with a cell-specific yield of 5200 virions/cells were achieved in bioreactor fed-batch mode. The PBG.PK-21 suspension cell line also shows potential to be used in a modern cell-based swine influenza vaccine production process for the veterinary market. Indeed, similar competitive virus titres (3.4 log(HAU/100µL)) and cell-specific yields were found for the swine influenza A/Bakum/1832/00 (H1N2) virus in batch mode. Finally, tests were also conducted regarding yellow fever virus production (live-attenuated 17DD YFV, RKI). In first scouting experiments, promising titres up to 3 x 106 PFU/mL were obtained in batch mode. First attempts towards process intensification using ATF-based perfusion systems with manually-adapted perfusion rate showed successful maintenance of cell-specific influenza A virus yields at high cell density. Titres up to 4 log(HAU/100µL) were obtained with 42 x 106 cells/mL. The glycosylation profile of influenza A/PR8/34 produced in PBG.PK-21 cell line was also analysed and will be compared to profiles of other cell lines. Overall, PBG.PK-21 suspension cells show a great potential to become the cell substrate of choice for many existing and new viral vaccine processes in next generation high-yield human as well as veterinary vaccine manufacturing. [1] Genzel, Y., et al., Vaccine, 2014. 32(24): p. 2770-81. [2] Tapia F, et al., PLoS ONE, 2017, 12(8): e0182553

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses

first_page settings Order Article Reprints Open AccessHypothesis Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses by Markus Kastner 1,†,‡, Andreas Karner 1,†,§ [ORCID] , Rong Zhu 1,† [ORCID] , Qiang Huang 2 [ORCID] , Andreas Geissner 3,4,‖, Anne Sadewasser 5,¶, Markus Lesch 6, Xenia Wörmann 6, Alexander Karlas 6,**, Peter H. Seeberger 3,4 [ORCID] , Thorsten Wolff 5 [ORCID] , Peter Hinterdorfer 1 [ORCID] , Andreas Herrmann 7 and Christian Sieben 8,9,* [ORCID] 1 Institute for Biophysics, Johannes Kepler University Linz, 4020 Linz, Austria 2 State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China 3 Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, 14476 Potsdam, Germany 4 Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany 5 Division of Influenza and other Respiratory Viruses, Robert Koch-Institute, 13353 Berlin, Germany 6 Molecular Biology Department, Max Planck Institute for Infection Biology, 10117 Berlin, Germany 7 Institut für Chemie und Biochemie, Freie Universität Berlin, Altensteinstraße 23a, 14195 Berlin, Germany 8 Nanoscale Infection Biology Group, Department of Cell Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany 9 Institute for Genetics, Technische Universität Braunschweig, 38106 Braunschweig, Germany * Author to whom correspondence should be addressed. † These authors contributed equally to this work. ‡ Current address: Materials Characterization Lab (MCL), Materials Research Institute (MRI), Pennsylvania State University, University Park, PA 16802, USA. § Current address: University of Applied Sciences Upper Austria, School of Medical Engineering and Applied Social Sciences, Garnisonstr. 21, 4020 Linz, Austria. ‖ Current address: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada. ¶ Current address: Secarna Pharmaceuticals GmbH & Co. KG, Am Klopferspitz 19, 82152 Planegg, Germany. ** Current address: Viral Vectors and Gene Therapeutics, ProBioGen AG, 13086 Berlin, Germany. Viruses 2023, 15(7), 1507; https://doi.org/10.3390/v15071507 Received: 9 May 2023 / Revised: 21 June 2023 / Accepted: 28 June 2023 / Published: 5 July 2023 (This article belongs to the Special Issue Physical Virology - Viruses at Multiple Levels of Complexity) Download Browse Figures Review Reports Versions Notes Abstract Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA’s receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus–cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA’s receptor binding preference does not necessarily reflect virus–cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells

Age and gender specific estimation of visceral adipose tissue amounts from radiological images in morbidly obese patients OPEN

Best predictions were found at intervertebral spaces L3-L4 for females (σ 5 = 688 ml, σ 1 = 832 ml) and L1-L2 for males (σ 5 = 846 ml, σ 1 = 992 ml), irrespective of age. In conclusion, VAT volumes in morbidly obese patients can be reliably predicted by multiplying the segmented VAT area at a gender-specific lumbar reference level with a fixed scaling factor and effective slice thickness. Obesity is a worldwide increasing healthcare problem. In the United States, for example, over two thirds of the adult population are either overweight (33%, BMI: 25-30 kg/m 2 ), obese (35%, 30-40 kg/m 2 ) or morbidly obese (6%, > 40 kg/m 2 ) 1 . While prevalence of obesity is still rising, in particular the morbid form 2 , more and more is known about its association with an increased overall mortality, often caused by cardiovascular diseases, diabetes or hypertension Quantification of abdominal VAT volumes by cross-sectional imaging, typically by computed tomography (CT) or magnetic resonance imaging (MRI), however, is generally time-consuming 7-9 . Various methods have already been proposed to estimate total VAT volumes from simple measurements on a limited number of slices. Studies using single or five slice VAT areas for VAT volume prediction have mainly focused on patients with BMI values below 40 kg/m 2 and data for the morbidly obese are lackin

Estimating steatosis and fibrosis: comparison of acoustic structure quantification with established techniques

To compare ultrasound-based acoustic structure quantification (ASQ) with established non-invasive techniques for grading and staging fatty liver disease

Estimating steatosis and fibrosis: comparison of acoustic structure quantification with established techniques

To compare ultrasound-based acoustic structure quantification (ASQ) with established non-invasive techniques for grading and staging fatty liver disease

Rab6 and Rab11 Regulate Chlamydia trachomatis Development and Golgin-84-Dependent Golgi Fragmentation

Many intracellular pathogens that replicate in special membrane bound compartments exploit cellular trafficking pathways by targeting small GTPases, including Rab proteins. Members of the Chlamydiaceae recruit a subset of Rab proteins to their inclusions, but the significance of these interactions is uncertain. Using RNA interference, we identified Rab6 and Rab11 as important regulators of Chlamydia infections. Depletion of either Rab6 or Rab11, but not the other Rab proteins tested, decreased the formation of infectious particles. We further examined the interplay between these Rab proteins and the Golgi matrix components golgin-84 and p115 with regard to Chlamydia-induced Golgi fragmentation. Silencing of the Rab proteins blocked Chlamydia-induced and golgin-84 knockdown-stimulated Golgi disruption, whereas Golgi fragmentation was unaffected in p115 depleted cells. Interestingly, p115-induced Golgi fragmentation could rescue Chlamydia propagation in Rab6 and Rab11 knockdown cells. Furthermore, transport of nutrients to Chlamydia, as monitored by BODIPY-Ceramide, was inhibited by Rab6 and Rab11 knockdown. Taken together, our results demonstrate that Rab6 and Rab11 are key regulators of Golgi stability and further support the notion that Chlamydia subverts Golgi structure to enhance its intracellular development

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: An individual patient data meta-analysis

Objective Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM-VCTE), Fibrosis-4 Index (FIB-4) and NAFLD (non-alcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. Design Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. Results Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs (<1.3; ≥2.67) followed by LSM-VCTE cut-offs (<8.0; ≥10.0 kPa) to rule-in or rule-out advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63-68) and 86% (84-87) with 33% needing a biopsy to establish a final diagnosis. FIB-4 cut-offs (<1.3; ≥3.48) followed by LSM cut-offs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37-39) and specificity of 90% (89-91) with 19% needing biopsy. Conclusion Sequential combinations of markers with a lower cut-off to rule-out advanced fibrosis and a higher cut-off to rule-in cirrhosis can reduce the need for liver biopsies