Insect cells platforms for fast production of Pseudo-Typed VLPs for drug and vaccine development
June 12-17, 2016
Expression systems capable of delivering high concentrations of membrane proteins in their native structure are essential in the vaccine field as well as in drug discovery. In this work, we took advantage of insect cell expression and site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to generate cell platforms for efficient production of membrane proteins on the surface of a protein scaffold, namely enveloped virus-like particles (VLPs). The expression of membrane proteins concomitantly with capsid proteins of enveloped viruses (e.g. HIV Gag or influenza M1) will enable their capturing in lipid rafts of the cellular plasma membrane and their display on the surface of budding VLPs, thus providing a native conformation for downstream assays.
Parental insect Sf-9 and High Five cells were randomly tagged with GFP-fused Gag or M1 proteins 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 observe that Gag localizes preferentially at the plasma membrane whereas M1 disperses within the cell. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well succeeded, allowing to recover cells tagged in loci supporting FMCE. We are currently evaluating the capability of both core proteins as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and Influenza HA proteins. For the latter, we will present recent results on the feasibility of combining stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs towards the development of an “universal” vaccine. This strategy surpasses standard methods for production of multivalent Influenza VLPs such as coinfections or the use of larger, unstable vectors.
Overall, modular insect cells platforms are being generated to be readily adaptable for production of a broad range of VLP-based vaccines as well as 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.
António Roldão, João Vidigal, Daniela Sequeira, Bárbara Fernandes, Mafalda M. Dias, Marco Patrone, Manuel JT Carrondo, Ana P. Teixeira, and Paula M. Alves, "Insect cells 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/28