TY - JOUR
T1 - Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins
AU - Suárez-Patiño, Sandra Fernanda
AU - Bernardino, Thaissa Consoni
AU - Núñez, Eutimio Gustavo Fernández
AU - Astray, Renato Mancini
AU - Pereira, Carlos Augusto
AU - Soares, Hugo R.
AU - Coroadinha, Ana S.
AU - Jorge, Soraia Attie Calil
PY - 2019/10/1
Y1 - 2019/10/1
N2 - The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.
AB - The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.
KW - BHK-21 cells
KW - Hepatitis C virus nonstructural protein 3 protease domain
KW - Rabies virus glycoprotein
KW - Semliki Forest Virus
KW - Serum-free medium
KW - Viral replicon particles
UR - http://www.scopus.com/inward/record.url?scp=85070803069&partnerID=8YFLogxK
U2 - 10.1007/s10616-019-00337-y
DO - 10.1007/s10616-019-00337-y
M3 - Article
AN - SCOPUS:85070803069
VL - 71
SP - 949
EP - 962
JO - Cytotechnology
JF - Cytotechnology
SN - 0920-9069
IS - 5
ER -
