A fluorescence protein toolbox for improving virus and virus-like particle-based biologics production in animal cells

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The demand for biologics is increasing at an unprecedented rate. As a result, the vaccine market is expected to increase from 24 billion dollars to 61 billion dollars by 2020 (Global Research – Center for Research Globalization). Viral threats on a global scale remain omnipresent and ever-changing. In the last two decades alone, four types of viruses have been the cause of major outbreaks: SARS coronavirus; avian and swine influenza viruses; Ebola virus; and most recently, Zika virus. Cell culture is the most attractive manufacturing platforms for rapid, scalable and controlled production of viral vaccines. This research program aims to bring substantial improvement to the production of viruses and virus-like particles (VLPs) in the Vero cell and insect cell (Sf9) platforms respectively, by developing a toolbox of well-characterized fluorescent protein (FPs) that can be used in both platforms. To improve the Vero platform, we aim to develop a single-cell suspension-adapted Vero line using a combination of cell culture conditions and gene knockdown strategies. We will also use our FP-toolbox to engineer combinations of FP that are responsive to the state of the cell to understand the effects of culture conditions and infection on the health of the cell. To improve the baculovirus expression vector – insect cell system, we aim to use the FP toolbox to gain insight on controlled expression of multiple proteins inside the cell. This control will be achieved by investigating alternative and novel promoter sequences to drive protein expression, as well as the use of CRISPR-Cas9 to modulate and downregulate protein expression. It is a goal of this program to increase the cell specific productivity of both of the platforms at increasing cell densities, and it is believed that the FP toolbox that will be developed will play an integral role in understanding changes in gene expression and state of the cell. This research program is geared towards training scientists and engineers in the control of biological products and systems. As a result, students get exposed to: manipulating DNA; working with bacteria (Escherichia coli); working with animal cell culture (insect and mammalian cells); working with viruses (baculovirus, influenza virus); utilizing bioreactors; purifying and characterizing biologics (viruses and VLPs); and creating mathematical models that help the analysis of the work. The skills that students will gain from this research program and environment are heavily sought by the bio-pharmaceutical industry, where there is a current need for highly qualified bioprocess/bio-engineers.