Baculoviruses for the production of virus-like particles
To expand the set of tools for modern vaccine design and production, it is necessary to develop robust strategies for the generation of recombinant proteins and bionanoparticles. For many viral diseases, classical whole virus vaccines are not suitable or don’t provide sufficient flexibility in terms of antigenic shift. The baculovirus expression vector system (BEVS) using insect cells has become an established large-scale production platform, increasingly used in the biopharmaceutical industry. Its popularity predominantly relates to large flexibility, serum-free suspension cultivation, lower production costs and substantial higher yields as compared to mammalian cells. It offers a wide range of post-translational modifications, proper protein folding and trafficking and has a long history of safe use.
Various recombinant virus-like particles (VLPs) have been manufactured using this system. HIV-1 Gag-based VLPs have shown to be a feasible and flexible platform for the expression of complex transmembrane glycoproteins. In pre-clinical studies, viral antigens such as the influenza virus A hemagglutinin have already proven to be highly immunogenic and effective when applied as particle vaccine, however, also non-viral proteins such as tumor antigens have been shown to induce protective anti-tumor immunity in this context.
As an engineering and production platform, the baculovirus/insect cell system offers the flexibility to specifically design and produce the antigen of choice within a short period of time. In combination with a robust insect cell line and a continuous production process, as well as optimized purification procedures, this system constitutes a flexible, fast and safe vaccine production platform contributing to overcome future challenges in health care.
Inducible stable expression of Adeno Associated viral components in HEK293 cells
In the context of the “CD-Laboratory for Knowledge-Based Production of Gene Therapy Vectors” lead by Prof. Astrid Dürauer (IBSE) we will establish various stable HEK cell lines, suitable for improved production of adeno associated viruses (AAV) for gene therapy. Adeno associated virus (AAV) particles are widely used in different gene therapy applications. Usually, they are produced by transient transfection of HEK293. Three plasmids are co-transfected: one expressing AAV Rep and Cap, the second expressing all essential helper factors from Adenovirus (AdV) and the third bearing the target gene. AdV helper factors E1B, E2A, E4 and VA are essential for the replication and efficient production of AAVs in HEK293 cells. E4 (together with E1B) regulates the expression of AAV genes by aiding in mRNA transport to the cytoplasm and support DNA replication by arresting the cell cycle in the G2 or S phase. To address the limited scalability and reproducibility in large scale production, in addition to the very high cost of plasmid production, the aim of this project is to generate and characterize different packaging and producer cell lines that contain Rep/Cap expressing genes and AdV helper protein expressing genes (E4, E2a and VA) in different combinations integrated into their genome under control of inducible and/or tunable promoters. Inducible systems will enable cells to grow unrestrained during expansion and allow for expression of the toxic helper proteins only during the transfection stage and production of gene therapy vectors. Tunable expression systems provide the advantage that fine-tuned optimization of expression levels of the different proteins and their combinations for most efficient virus production is possible. Further, these cell lines can be used to test other optimization strategies with a given background of viral gene expression levels.
The force of sugar in the SARS-CoV-2 spike/ACE-2 interaction
(FWF P35166)
Virus–receptor interactions are pivotal in establishing an infection. The SARS-CoV-2 spike protein and the human receptor ACE2 are both heavily glycosylated and glycans indirectly support or are directly involved in the interaction of the two proteins. As they might have a dramatic impact on viral transmissibility and infection, any spike mutation or ACE2 single nucleotide polymorphism that results in the loss of a strategically-positioned glycan in or close to the binding interface deserve our attention.
Together with our co-operation partner Peter Hinterdorfer (Johannes Kepler University, Linz) we aim to gain a profound understanding of the role of glycans in the SARS-CoV-2 spike – ACE2 interaction. Hereby, we focus on site-specific ablations of glycosylation sites within the binding interface, most notably on naturally occurring ACE-2 glycovariant polymorphisms. We will evaluate the capability of the interaction partners to bind under dynamic conditions and quantify binding strengths and kinetics, as well as map their interaction energy landscape. In addition, we will monitor the involvement of specific glycans in modulating the proteins’ conformational dynamics and test the inhibitory effect of soluble ACE-2 glycovariants on the binding of the spike to cellular ACE-2 receptors. Our comprehensive investigations will not only result in a valuable collection of data for deciphering the mechanisms of spike-ACE-2 variant interaction, but also provide an experimental basis for the design of novel therapeutics for effective blocking of viral variant entry.
This project is funded by the Austrian Science Fund.
Rapid detection of SARS-CoV-2 with a novel electronic bio-sensor – an alternative to cell culture for the determination of infectivity and an early tool for the identification of SARS-CoV-2 variants
(FWF P35103)
Currently available Point-of-care (POC) devices permit the diagnosis of an acute SARS-CoV-2 infection but do neither allow to discriminate between viral variants, nor do they indicate whether a specimen contains infectious virus. A diagnostic platform tackling all these three questions could be extremely helpful in the fight against the SARS-CoV-2 pandemic, while saving precious health system resources and avoiding unnecessarily long quarantine for patients.
Together with our co-operation partners Robert Strassl (Medical University of Vienna) and Patrik Aspermair (Austrian Institute of Technology, Tulln) we work on innovative SARS-CoV-2 detection systems for the rapid detection of SARS-CoV-2 from respiratory specimens with the capability of determining the SARS-CoV-2 variant as well as the infectivity status of positive COVID-19 samples. Hereby, a combination of bio-sensor surfaces in an electronic device will be generated, referenced by an optical measurement tool., this should allow for dual-sensing of viral genetic material as well as viral proteins. Pre-validation of different biosensor surfaces will be done under minimal containment facilities (BSL-1) using innovative surrogate viruses/virus-like particles (VLPs) as test analytes and will then be validated with clinical specimens covering different viral variants and varying degrees of infectiousness (viral culture, Ct values).
This project is funded by the Austrian Science Fund.
Extracellular vesicles and their role within the baculovirus/ insect cell expression system
Virtually any type of eukaryotic cell releases different kinds of small particles altogether referred to as extracellular vesicles (EVs). Those EVs contain specific proteins, nucleic acids and lipids and are used to confer information intracellularly. EVs have frequently been studied in mammalian cells where it has been shown that viral infection can alter EV amount, size distribution as well as composition, contributing to either further spreading or containment of the infection. As the baculovirus/ insect cell expression system is a viral production system, this strongly indicates that EVs could play a major role in the system’s performance. Yet until now, the role of EVs within the baculovirus/ insect cell expression system has not been elucidated.
We want to investigate how baculovirus infection of insect cells modifies the release of EVs. EVs are purified from infected as well as non-infected cells and changes in distribution and content are analyzed. Further, we examine the impact of enrichment as well as depletion of EVs. Overall, we want to shed light on EV-based intracellular communication within the baculovirus/ insect cell expression system and generate new tools for process control to further improve biopharmaceutical production in this expression system.
Optimization of Bacillus strains for improved protein expression/ secretion
Bacillus subtilis is a Gram-positive, spore-forming soil bacterium, which due to its GRAS (Generally Recognized as Safe) status and its use in the production of various foods is considered very safe and therefore represents an interesting cell factory. In addition, B. subtilis expresses a large number of different enzymes with a wide range of different substrate specificities. Many of these homologous enzymes are very efficiently secreted into the environment by the bacterium and can therefore also be used commercially on a large scale, e.g. be used in washing powder or in the paper industry. Unfortunately, the expression and secretion of heterologous proteins is usually less efficient because some bottlenecks such as incorrect protein folding, inefficient targeting to the secretion apparatus and degradation of the target proteins by host proteases interfere with recombinant protein production.
The aim of this project is to use current genetic engineering methods to eliminate these bottlenecks in B. subtilis and to provide a powerful expression system for biotechnologically relevant proteins. In addition to B. subtilis, other B.spp. with similar positive properties will be tested for their suitability as a cell factory.
Bioremediation of per- and polyfluoroalkyl substances (PFAS biorem)
PFAS are persistent organic compounds that have been widely used since the 1940s in industrial and commercial products, such as fire-fighting foams, cookware materials, high-temperature lubricants, ski wax, water-repellent clothing and many other products. They are now detectable globally in the environment, contaminating soil and water and potentially harming human health. Although PFAS are known for their high resistance to biodegradation, there are some species that can at least partially degrade these compounds, e.g. some Pseudomonas spp. In our project, we will use an adapted variant of the method described by Luan et al. (2013) to provide Pseudomonas mutants with improved PFAS degradation capabilities. We will generate plasmids encoding mutant variants of the protein DnaQ (corresponding to the ε-subunit of DNA polymerase III); DnaQ is responsible for the proofreading activity of the DNA polymerase III holoenzyme. Depending on the DnaQ variant, plasmids with strong, medium and weak mutator properties will be obtained. Pseudomonas spp. Will be transformed with these plasmids and cultivated under conditions that induce the expression of mutated DnaQ variants, leading to increased mutation rates. Growth in the presence of PFAS in the culture medium leads to adaptation through enhanced enzyme sets/degradation pathways/utilization of PFAS in specific clones that can be identified and isolated by high-throughput screening methods. Strains with the best PFAS degradation capabilities in the culture medium will then be tested for their PFAS-reducing abilities in different environmental samples such as water or soil. In addition to standardized quantitative methods, specific LC-HRMS/MS and LC-ion mobility-HRMS/MS based analytical methods will be developed and adapted at the Institute of Chemistry (AG Prof. Dr. Stephan Hann) to characterize the degradation and transformation products generated during PFAS degradation.
This project is funded by the Federal Ministry for Climate Action, Environment, Energy, Mobility, Innovation and Technology.
REMBAC-cassette, a rapid, efficient and manifold BacMam tool for recombinant protein expression
Efficient recombinant protein production requires stable cell lines or often relies on inefficient transfection processes. Baculoviral transduction of mammalian cells (BacMam) offers cost-effective and robust gene transfer and straightforward scalability. The advantages over conventional approaches are, no need of high biosafety level laboratories, efficient transduction of various cell types and transfer of large transgenes into host cells. We developed high-level expression cassettes supporting high efficiency baculoviral transduction and high-level recombinant protein expression. This REMBAC-cassette displayed significantly higher transduction efficiencies and expression levels compared to conventional transfection in suspension and adherent cells (HEK, CHO, Vero). Irrespective of the cell line, transduction reached nearly 100% efficiency and led to almost 10-fold increases of gene expression levels. These results are also valid for larger scale production processes in a batch and a fed-batch manner. The versatility of the cassette allows for expression of different soluble proteins with high degrees of complexity. The REMBAC-cassette incorporated into the BacMam platform is a manifold tool offering advantages over standard transfection, in the scalability, efficiency and gene expression, which results in higher yields, shorter cultivation times and consequently cost-effective production processes.
REMBAC (Rapid Efficient Manifold Baculovirus Transduction) for stable cell line development
We have developed a novel system for efficient transduction of cells using a baculovirus vector and permitting site-specific genome integration. This system – termed REMBAC (rapid efficient manifold baculovirus transduction) - is to be used for stable cell line development (SCLD), with applications ranging from the expression of multiprotein assemblies, including virus-like particle (VLP) vaccines, adenovirus-associated vectors (AAVs) for gene therapy or antibodies as therapeutics or for R&D. The system facilitates footprint-free site-specific genome integration in combination with the modular REMBAC cassette into nearly all mammalian cell types. Since complex products of particulate structure or gene therapy vectors are conventionally expressed from two or more plasmids, the incorporation of the REMBAC cassette into the baculovirus allows simultaneous expression of all components at tailored levels. In contrast to most cell transfection reagents, baculovirus transduction does not affect cell viability, supporting a high-quality cell pool to start from. Moreover, the high versatility of the system is permitted by the baculovirus surface glycoprotein GP64, which mediates entry into broad range of mammalian cell types, irrespective of the cell cycle status, which is not achievable with conventional lentiviral or retroviral approaches.
