DEVELOPMENT OF INSECT CELL PLATFORMS FOR FAST

DEVELOPMENT OF INSECT CELL PLATFORMS FOR FAST PRODUCTION OF PSEUDO-TYPED VLPS
FOR DRUG AND VACCINE DEVELOPMENT
João Vidigal, IBET, Instituto de Biologia Experimental e Tecnológica ; Instituto de Tecnologia Química e
Biológica António Xavier, Universidade Nova de Lisboa, Portugal
[email protected]
Bárbara Fernandes, IBET, Instituto de Biologia Experimental e Tecnológica/ Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa
Mafalda M Dias, IBET, Instituto de Biologia Experimental e Tecnológica ; Instituto de Tecnologia Química e
Biológica António Xavier, Universidade Nova de Lisboa, Portugal.
Marco Patrone, Biocrystallography Unit, San Raffaele Scientific Institute, Italy.
Manuel JT Carrondo, IBET, Instituto de Biologia Experimental e Tecnológica ; Instituto de Tecnologia Química e
Biológica António Xavier, Universidade Nova de Lisboa, Portugal.
Paula M Alves, IBET, Instituto de Biologia Experimental e Tecnológica ; Instituto de Tecnologia Química e
Biológica António Xavier, Universidade Nova de Lisboa, Portugal.
Ana P Teixeira, IBET, Instituto de Biologia Experimental e Tecnológica ; Instituto de Tecnologia Química e
Biológica António Xavier, Universidade Nova de Lisboa, Portugal.
Key Words:
Vaccines.
Stable insect cell platform; Recombinase-mediated cassette exchange; HIV Gag-VLPs; GPCRs;
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 wellsucceeded (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.