Process development for a flexible vaccine vector platform based on recombinant life virus

Vaccines are one of the most important, safe and efficient interventions to protect people from illness, disability and death. In recent years several new viral outbreaks where no vaccines are currently available were reported worldwide. Therefore, the development of flexible processes for the production of vaccines is urgently needed. This project aims at developing a platform process for the production of different viral vaccines. The core technology is based on the fact that large recombinant genes coding for selected, foreign antigens can be inserted into the genome of a well-established virus vaccination vector. The vaccine delivers the selected antigens directly to macrophages and dendritic cells, the most potent and effective antigen-presenting cells, thereby triggering a specific immune response to the selected antigens. As a replicating vector, the vaccine continuously expresses antigens even after immunization. This setup results in a powerful, antigen-focused immune response, which is expected to confer long-term immunity as shown for the measles vaccine. The challenges in production process design for such a vaccine are the establishment of a robust cell expansion and infection strategy as well the development of efficient downstream processing methods including several chromatography principals, ultra-diafiltration and employment of bio recognition principles. The implementation of a meaningful real-time process monitoring/characterization concept furthermore serves as a basis for reliable in-process control strategies (e.g. the prediction of the optimal infection/harvesting time point)

Jerusalem, Israel/Palästina. Die Grabung auf dem Zionsberg im Südwesten der Altstadt von Jerusalem (im Bereich des historischen Anglikanisch-Preußischen Friedhofs)

The area in the south of Mt. Zion lies within the borders of the Protestant Cemetery. Two English pioneers, F. Bliss and A. Dickie, followed the course of the ancient city walls at the end of the 19th century by digging in subterranean tunnels and discovered that there are three walls above each other in this area. The most recently constructed one is from the first half of the 5th century. An older one with the same outline as the aforementioned is the one that Flavius Josephus refers to in Jewish Antiquities and the oldest one belongs to the Iron Age. This last wall might be a rare example of the wall that Hiskijahu built as he enlarged the city to create a protected space for the refugees from the northern Kingdom of Israel at the end of the 8th century BC. In addition to Bliss and Dickie, another excavation was begun in the same area by father B. Pixner in the 1980s. Y. Zelinger, who works for the IAA, then undertook a more focused excavation in an area near the cemetery that shows the continuation of the same city walls. His results allow a partial reconsideration of the findings in the cemetery. The GPIA started its work at this exceptional and unstudied site of ancient Jerusalem in 2015. In a first season, the old excavations were carefully cleaned and documented after more than 20 years of neglect. The work concentrated on the city walls as well as on the so-called “Essene Gate” which is also mentioned by Flavius Josephus. In 2016 the project was enlarged and three large new squares were opened. These squares are in the inside of the ancient city of Jerusalem; the aim of the project is to study the city’s settlement structures. After digging through banking structures from the Modern, Islamic and Medieval periods, the excavation has now reached the Byzantine level. Many promising loci have been documented that lead into the city extension built during the time of the influential empress Eudokia in the first half of the 5th century. The upcoming seasons are expected to reveal not only fascinating Byzantine structures but monumental Hasmonean/Herodian buildings. Since there are still three meters of cultural debris to the bedrock, also the traces of the Iron Age city can be awaited in that area. The GPIA’s Mt. Zion Excavation project offers the great and rare opportunity to research the history of Jerusalem in a huge and so far untouched area directly related to the old city walls.

Process development for a flexible vaccine vector platform based on recombinant life virus

Vaccines are one of the most important, safe and efficient interventions to protect people from illness, disability and death. In recent years several new viral outbreaks where no vaccines are currently available were reported worldwide. Therefore, the development of flexible processes for the production of vaccines is urgently needed. This project aims at developing a platform process for the production of different viral vaccines. The core technology is based on the fact that large recombinant genes coding for selected, foreign antigens can be inserted into the genome of a well-established virus vaccination vector. The vaccine delivers the selected antigens directly to macrophages and dendritic cells, the most potent and effective antigen-presenting cells, thereby triggering a specific immune response to the selected antigens. As a replicating vector, the vaccine continuously expresses antigens even after immunization. This setup results in a powerful, antigen-focused immune response, which is expected to confer long-term immunity. The challenges in production process design for such a vaccine are the establishment of a robust cell expansion and infection strategy as well the development of efficient downstream processing methods including several chromatography principals, ultra-diafiltration and employment of bio recognition principles. The implementation of a meaningful monitoring/characterization concept furthermore serves as a basis for reliable in-process control strategies

Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Generation of recombinant influenza A derived ribonucleoprotein complexes

Influenza A basierende Ribonucleoproteincomplexe (RNP) bestehen aus negativ-strängiger RNA, die von Nucleoprotein (NP) umgeben ist. Komplementäre Regionen an den 5' und 3' Enden können sich zu einer Duplexstruktur zusammenlagern, was von essentieller Bedeutung für Transkription und Replikation ist. Diese werden von einer heterotrimeren Polymerase, bestehend aus PA, PB1 und PB2 Subeinheiten, bewerkstelligt, welche an die Duplexstruktur der viralen RNA gebunden ist. Auf Grund der Tatsache das Influenza A RNP Komplexe die minimal nötigen infektiösen Partikel darstellen, wird ihre Verwendung für in vitro / in vivo Gene delivery und für die Generierung rekombinanter Viren angedacht. Eine weitere Verwendungsmöglichkeit besteht im Bereich der Impfstoffentwicklung. Das Ziel dieser Arbeit war es die 4 RNP-Proteine unter zur Hilfenahme von Baculoviren rekombinant in einem Insektenzellsystem zu exprimieren. Wir haben damit begonnen die für die RNP Bildung ideale MOI / hpi Kombination zu ermitteln und weiters versucht die Komplexe mittels Blue Native Page in ihrem nativen Zustand sichtbar zu machen. Im nächsten Schritt isolierten wir nukleäre RNP Komplexe mittels Ultracentrifugation und bestätigten deren Anwesenheit in der Pellet-Fraktion mittels Western Blot. Um die Funktionalität der viralen Polymerasen zu zeigen, wurden Sf9 Zellen mit Baculoviren die die 4 RNP-Proteine exprimieren, als auch mit Baculoviren die negativ-strängige RNA (kodiert für ein GFP Fusionsprotein) zur Verfügung stellt, infiziert. Wenn nun die Polymerasen aktiv sind, kommt es zur Expression von GFP. Um in weitere Folge die Aktivität der Polymerasen zu testen, wurden Sf9 Zellen mit Baculoviren, die RNP-Proteine codieren, infiziert, mit in vitro transkripeirter RNA transfiziert und auf GFP-Expression hin beobachtet. Die Korrektheit der in vitro transkriperten RNA konnte durch Infektion von MDCK und CHO Zellen mit dem Influenza A Stamm PR/8/34 und anschleißender RNA Transfektion gezeigt werden.The influenza A derived ribonucleoprotein (RNP) complexes consist of negative single stranded RNA covered with Nucleoprotein (NP). Complementary regions at the 5' and 3' termini can anneal to form a bulged duplex structure that is essential for transcription and replication, which is achieved by a heterotrimeric polymerase complex, consisting of PA, PB1 and PB2. The polymerase is found to be tightly associated with vRNA 5' and 3' termini. Due to the fact that RNP complexes are the minimal infectious particle of influenza A virus, they are intended for vitro / in vivo gene delivery, as well as for generation of recombinant virus. Another application is the use of RNP complexes in vaccine design. The goal of this study was to recombinantly express the 4 RNP proteins in an insect cell system, using baculovirus for gene delivery. We first established MOI / hpi combinations ideal for RNP formation in the nucleus and tried to visualize complexes in their native state, which was achieved by Blue Native page. We further attempted to isolate RNPs by velocity sedimentation and confirmed the presence of nucleoprotein in pellet fraction by western blot. To show functionality, insect cells were infected with baculovirus delivering RNP proteins together with baculovirus delivering negative single-stranded RNA coding for a GFP fusion protein. If polymerases work, negative sense RNA should be transcribed to mRNA, leading to GFP expression. For testing the activity of viral polymerases in an insect cell system, Sf9 cells were transfected with in vitro transcribed negative-stranded RNA and monitored for GFP expression. For further verifying in vitro transcribed RNA we infected CHO and MDCK cells with the influenza A strain PR/8/34 and transfected the cells with RNA. We finally observed 4 GFP expressing CHO cells leading to the assumption that in vitro transcribed RNA was correct.Palmberger DieterWien, Univ. für Bodenkultur, Masterarb., 2007OeBB(VLID)103655

Insect cells as an engineering platform for the production of antibodies and antibody derived products

In den letzten Jahren ist es zu einer deutlich gestiegenen Nachfrage an neuen therapeutischen Antikörper-Produkten gekommen. Die Entwicklung neuer Design- und Selektionsplatformen, sowie geeigneter Produktionssysteme ist somit von großer Bedeutung. Um die Eigenschaften dieser neu gestalteten Fragmente im Detail bewerten zu können haben wir ein effizientes Selektionsverfahren entwickelt. Hierfür wurden Baculovirus-Vektoren generiert die es ermöglichen ein IgG1 Fc-Fragment, welches an die Transmembran-Region der Influenza A Neuraminidase fusioniert wurde, um auf der Oberfläche von Insekten- und Säugerzellen zu präsentieren. Zelluläre Expression wurde mittels Durchflusszytometrie bestätigt, eine korrekte Faltung und Dimerbildung wurde durch Bindung an Staphylococcus aureus Protein A (SpA) und den humanen FcRI getestet. Weiters wurde ein auf Inseltenzellen basierendes System für die Produktion von human-ähnlichen Antikörpern entwickelt. Wir haben den humanen anti-gp41 Antikörper 3D6 in verschiedenen Insektenzellen exprimiert und in Bezug auf Produktausbeute, Spezifität und Glykosylierungsmuster mit einem in CHO exprimierten 3D6 Antikörper verglichen. "High Five" Zellen zeigten eine Ausbeute vergleichbar mit jener aus transienten Expressionen in Säugerzellen. Wir bestimmten die an Asparagin-297 gebundenen Oligosaccharid Strukturen der schweren Kette und testeten deren Funktionalität in Bezug auf Antigen-Bindung und ihre Fähigkeit Effektor-Funktionen auszulösen. "High Five" Zellen zeigten hierbei ein Glykosylierungsmuster das auf eine reduzierte biologische Aktivität vermuten lässt. Deshalb entwickelten wir ein Glykosylierung-Modul welches eine flexible Modifizierung der Glykosylierungskapazität diverser Insektenzellinien ermöglicht.In recent years there has been an increase in both availability and demand for new therapeutic antibody products. Thus, novel engineering and screening platforms as well as efficient and suitable expression schemes are required. In order to evaluate the specificities of newly designed antibody derived fragments we established an efficient selection method. Baculovirus vectors were designed to express and display an IgG1 Fc-fragment fused the transmembrane region of the Influenza A virus neuraminidase on the surface of insect and mammalian cells. Cellular expression was confirmed by FACS analysis; proper folding and dimer formation was tested by binding to Staphylococcus aureus Protein A (SpA) and human FcRI respectively. Further, an efficient insect cell based expression system was evaluated and established to produce human-like antibodies. We expressed the human anti-gp41 antibody 3D6 in different insect cells and compared product yield, specificity and glycosylation patterns to a 3D6 antibody expressed in Chinese hamster ovary cells. Using “High Five” cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG1 heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. “High Five” cells showed a glycosylation pattern that might lead to a reduced biological activity. We therefore established a glycosylation module giving the possibility to flexibly modify the glycosylation capability of different insect cell lines.Dieter PalmbergerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2011OeBB(VLID)193150

SweetBac: A New Approach for the Production of Mammalianised Glycoproteins in Insect Cells

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine b1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showe

Development of a Dual-Vector System Utilizing MicroRNA Mimics of the <i>Autographa californica</i> miR-1 for an Inducible Knockdown in Insect Cells

The baculovirus-insect cell expression system is a popular tool for the manufacturing of various attractive recombinant products. Over the years, several attempts have been made to engineer and further improve this production platform by targeting host or baculoviral genes by RNA interference. In this study, an inducible knockdown system was established in insect (Sf9) cells by combining an artificial microRNA precursor mimic of baculoviral origin and the bacteriophage T7 transcription machinery. Four structurally different artificial precursor constructs were created and tested in a screening assay. The most efficient artificial microRNA construct resulted in a 69% reduction in the fluorescence intensity of the target enhanced yellow fluorescent protein (eYFP). Next, recombinant baculoviruses were created carrying either the selected artificial precursor mimic under the transcriptional control of the T7 promoter or solely the T7 RNA polymerase under a baculoviral promoter. Upon co-infecting Sf9 cells with these two viruses, the fluorescence intensity of eYFP was suppressed by ~30&#8315;40% on the protein level. The reduction in the target mRNA level was demonstrated with real-time quantitative PCR. The presented inducible knockdown system may serve as an important and valuable tool for basic baculovirus-insect cell research and for the improvement of production processes using this platform