Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

Autor(es)

Suarez-Patino, Sandra Fernanda ^[1] ; Bernardino, Thaissa Consoni ^[1] ; Fernandez Nunez, Eutimio Gustavo ^[2] ; Astray, Renato Mancini ^[1] ; Pereira, Carlos Augusto ^[1] ; Soares, Hugo R. ^{[3, 4]} ; Coroadinha, Ana S. ^{[3, 4]} ; Calil Jorge, Soraia Attie ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Imunol Viral, Ave Vital Brasil 1500, BR-05503900 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Grp Engn Bioquim, EACH, Sao Paulo - Brazil

^[3] IBET, Apartado 12, Oeiras - Portugal

^[4] Univ Nova Lisboa, Inst Tecnol Quim & Biol Antonio Xavier, Ave Republ, Oeiras - Portugal

• Tipo de documento: Artigo Científico
• Fonte: Cytotechnology; v. 71, n. 5, p. 949-962, OCT 2019.
• Citações Web of Science: 0

Resumo

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. (AU)

• Processo FAPESP: 11/05807-4 - Estudo da adaptação de células de mamíferos em meios de cultura livre de soro para otimizar a produção de proteínas recombinantes
• Beneficiário: Soraia Attie Calil Jorge
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/00978-8 - Produção de proteínas recombinantes em células bhk-21 cultivadas em meio livre de soro fetal bovino
• Beneficiário: Sandra Fernanda Suárez Patiño
• Modalidade de apoio: Bolsas no Brasil - Doutorado