title: Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles.
abstract: Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Avenida da República, 2780-157 Oeiras, Portugal.|iBET, Instituto de Biologia Experimental e Tecnológica , Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Sofia B
authors.initials: SB
authors.lastname: Carvalho
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa , Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors.forename: João M
authors.initials: JM
authors.lastname: Freire
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Avenida da República, 2780-157 Oeiras, Portugal.|iBET, Instituto de Biologia Experimental e Tecnológica , Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Mafalda G
authors.initials: MG
authors.lastname: Moleirinho
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Avenida da República, 2780-157 Oeiras, Portugal.|iBET, Instituto de Biologia Experimental e Tecnológica , Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Francisca
authors.initials: F
authors.lastname: Monteiro
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa , Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Gaspar
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa , Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors.forename: Miguel A R B
authors.initials: MARB
authors.lastname: Castanho
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica , Apartado 12, 2780-901 Oeiras, Portugal.|Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , 2829-516 Monte da Caparica, Portugal.
authors.forename: Manuel J T
authors.initials: MJT
authors.lastname: Carrondo
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Avenida da República, 2780-157 Oeiras, Portugal.|iBET, Instituto de Biologia Experimental e Tecnológica , Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa , Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.|Department of Chemistry, University of Cambridge , Lensfield Road, CB2 1EW Cambridge, United Kingdom.
authors.forename: Gonçalo J L
authors.initials: GJL
authors.lastname: Bernardes
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Avenida da República, 2780-157 Oeiras, Portugal.|iBET, Instituto de Biologia Experimental e Tecnológica , Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Peixoto