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.
VLPs as versatile tool to study influenza-specific immunity in the context of postinfluenza bacterial infections
(FWF I3490-B30, 06/2018 – 05/2021)
Influenza virus infections pre-dispose an individual for secondary bacterial infections. Such secondary infections generally result in more severe diseases and are believed to play a significant role in influenza-associated mortality, especially when there is little or no pre-existing immunity towards the preceding viral strain (such as in an influenza pandemic). In addition to the influenza hemagglutinin, several other viral proteins have been identified as key mediators in increasing susceptibility of an influenza-infected host to bacterial infection. However, these proteins are either not standardized or not present in current influenza vaccines.
In an ongoing FWF/RSF-funded bilateral joint project (project partner: Lab Irina A Levena, I. Mechnikov Research Institute of Vaccines and Sera, Moscow) we aim to elucidate the role of immunity towards these underinvestigated influenza antigens in the context of postinfluenza bacterial infections in the mouse model. To do so, we incorporate relevant influenza antigens in insect-expressed HIV-1 Gag VLPs – either as surface-displayed and/or internal cargo – and use them as highly immunogenic tool to study how immunity towards these antigens alone or in combination may help in the effective control of such multi-infection scenarios.
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.