TY - JOUR
T1 - Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production
AU - Sequeira, Daniela P.
AU - Correia, Ricardo
AU - Carrondo, Manuel J.T.
AU - Roldão, António
AU - Teixeira, Ana P.
AU - Alves, Paula M.
N1 - info:eu-repo/grantAgreement/FCT/3599-PPCDT/134566/PT#
"EDUFLUVAC" (FP7-HEALTH-2013-INNOVATION-1, GA n. 602640.
"Investigador FCT" Program (IF/01704/2014).
IF/01704/2014/CP1229/CT0001).
Sem PDF conforme despacho.
PY - 2018/5/24
Y1 - 2018/5/24
N2 - 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 × 106 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2 × 106 cells/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 × 106 cells/mL showed comparable HA titers per cell to those of CCI of 2 × 106 cells/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 2 L 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.
AB - 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 × 106 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2 × 106 cells/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 × 106 cells/mL showed comparable HA titers per cell to those of CCI of 2 × 106 cells/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 2 L 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.
KW - Bioprocess optimization
KW - Hemagglutinin-based vaccine
KW - Modular strategy
KW - Multivalent influenza virus-like particles
KW - Scale-up
KW - Stable insect cells
UR - http://www.scopus.com/inward/record.url?scp=85014803559&partnerID=8YFLogxK
U2 - 10.1016/j.vaccine.2017.02.043
DO - 10.1016/j.vaccine.2017.02.043
M3 - Article
C2 - 28291648
AN - SCOPUS:85014803559
VL - 36
SP - 3112
EP - 3123
JO - Vaccine
JF - Vaccine
SN - 0264-410X
IS - 22(SI)
T2 - 6th Conference of Vaccine Technology
Y2 - 12 June 2016 through 17 June 2016
ER -