title: Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.
abstract: Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×106cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×106cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×106cells/mL showed comparable HA titers per cell to those of CCI of 2×106cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable with baculovirus-mediated expression in insect cells as an efficient platform for production of multi-HA influenza VLPs, surpassing the drawbacks of traditional co-infection strategies and/or the use of larger, unstable vectors.
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; ITQB NOVA-Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal.
authors.forename: Daniela P
authors.initials: DP
authors.lastname: Sequeira
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; ITQB NOVA-Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Correia
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: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; ITQB NOVA-Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal. Electronic address: aroldao@ibet.pt.
authors.forename: António
authors.initials: A
authors.lastname: Roldão
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; ITQB NOVA-Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal. Electronic address: anat@ibet.pt.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Teixeira
authors.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; ITQB NOVA-Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D019149:Bioreactors; D018929:Cell Culture Techniques; D002460:Cell Line; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D007252:Influenza Vaccines / Q000096:biosynthesis* / Q000276:immunology; D007313:Insecta / Q000166:cytology; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D058425:Vaccines, Virus-Like Particle
keywords: Bioprocess optimization; Hemagglutinin-based vaccine; Modular strategy; Multivalent influenza virus-like particles; Scale-up; Stable insect cells