Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins
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.
KeywordsBHK-21 cells Hepatitis C virus nonstructural protein 3 protease domain Rabies virus glycoprotein Serum-free medium Semliki Forest Virus Viral replicon particles
Viral replicon particles of Semliki Forest Virus
BHK-21 cells grown in DMEM supplemented with 10% of fetal bovine serum
BHK-21 cells adapted to serum free medium
- VRP-GFP-SFM or VRP-GFP-FBS
Viral replicon particle carrying the GFP gene produced in BHK/SFM or BHK/FBS respectively
- VRP-NS3p-SFM or VRP-NS3p-FBS
Viral replicon particle carrying the NS3p protease domain gene produced in BHK/SFM or BHK/FBS, respectively
- VRP-RVGP-SFM or VRP-RVGP-FBS
Viral replicon particle carrying the RVGP gene produced in BHK/SFM or BHK/FBS
This work was financially supported by grants from FAPESP- Fundação de Amparo à Pesquisa do Estado de São Paulo (2011/05807-4), CNPq- Conselho Nacional de Desenvolvimento Científico e Tecnológico (152538/2012-7) and Butantan Foundation. Carlos Augusto Pereira was the recipient of a CNPq 1A senior fellowship. Sandra F Suárez-Patiño had scholarships from FAPESP (2012/00978-8). Ph.D. Ana Lia Pradella Puglia, Ph.D. Alexandre Goncalves Rezende, Ph.D. Jorge Mario Da Costa Ferreira Junior and Bs.C. Vera Lúcia Boldorini for scientific and technical assistance.
Compliance with ethical standards
Conflict of interests
The authors declare that they have no conflict of interests.
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