Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics

Marco Silvano; Nikolaus Virgolini; Ricardo Correia; Colin Clarke; Inês A Isidro; Paula M Alves; António Roldão

doi:10.3389/fbioe.2023.1143255

Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics

Front Bioeng Biotechnol. 2023 Mar 6:11:1143255. doi: 10.3389/fbioe.2023.1143255. eCollection 2023.

Authors

Marco Silvano^{1

2}, Nikolaus Virgolini^{1

2}, Ricardo Correia^{1

2}, Colin Clarke^{3

4}, Inês A Isidro^{1

2}, Paula M Alves^{1

2}, António Roldão^{1

2}

Affiliations

¹ iBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
² ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
³ NIBRT-National Institute for Bioprocessing Research and Training, Dublin, Ireland.
⁴ School of Chemical and Bioprocess Engineering, University College Dublin, Dublin, Ireland.

Abstract

The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization.

Keywords: IC-BEVS; cell heterogeneity; influenza VLP; pathway analysis; single-cell RNA sequencing.

Grants and funding

This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 813453 and the grant agreement N° 730964, and by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through the following initiatives: “Investigador FCT” Program (IF/01704/2014), Exploratory Research and Development Project (IF/01704/2014/CP1229/CT0001), PhD fellowships (SFRH/BD/134107/2017), iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Funding from INTERFACE Programme, through the Innovation, Technology, and Circular Economy Fund (FITEC), is gratefully acknowledged.