Development of insect cell platforms for fast production of pseudo-typed VLPs for drug and vaccine development
June 12-17, 2016
Production technologies providing high concentrations of membrane proteins in their native structure are essential in the vaccine field, as well as to support the drug discovery pipeline. In this work, we took advantage of insect cell expression and site specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to develop cell platforms for efficient production of membrane proteins on the surface of enveloped virus-like particles (VLPs). The co-expression of membrane proteins with capsid proteins of enveloped viruses (such as HIV Gag) will enable their capturing in lipid rafts of the cellular plasma membrane and displaying on the surface of budding VLPs, thus providing a native conformation for downstream assays. Parental Sf9 and Hi5 insect cells were randomly tagged with a GFP-fused Gag protein and FACS enriched with cells tagged in genomic “hot-spots” supporting high expression. A linker including a Flp recognition target (FRT) site was used to allow posterior removal of the marker gene from the particle through cassette exchange. By confocal microscopy we could see that Gag localizes preferentially at the plasma membrane, whereas by electron microscopy we could detect correctly assembled Gag-VLPs in the culture supernatant of both cell hosts. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well-succeeded (showing that the FRT site composing the linker fusing the two genes does not impact recombination), allowing to recover cells tagged in loci supporting FMCE. We are currently evaluating the capability of the Gag-VLPs as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and Influenza HA proteins. Overall, modular insect platforms are being constructed to be readily adaptable to produce a broad range of VLP-based vaccines and receptor display particles for drug screening or antibody discovery.
Acknowledgments: Funding from European Commission (Project EDUFLUVAC; Grant nr. 602640) and Fundação para a Ciência e a Tecnologia through the project EXPL/BBB-BIO/1541/2013 and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012.
João Vidigal, Bárbara Fernandes, Mafalda M. Dias, Manuel JT Carrondo, Paula M. Alves, Ana P. Teixeira, and Marco Patrone, "Development of insect cell platforms for fast production of pseudo-typed VLPs for drug and vaccine development" in "Vaccine Technology VI", Laura Palomares, UNAM, Mexico Manon Cox, Protein Sciences Corporation, USA Tarit Mukhopadhyay, University College London, UK Nathalie Garçon, BIOASTER Technology Research Institute, FR Eds, ECI Symposium Series, (2016). http://dc.engconfintl.org/vaccine_vi/89