Intracellular Delivery of HCV NS3p gene using vectored particles

Lemos, Marcos Alexandre Nobre; Patiño, Sandra Fernanda Suárez; Bernardino, Thaissa Consoni; Coroadinha, Ana S.; Soares, Hugo R.; Astray, Renato Mancini; Pereira, Carlos Augusto; Jorge, Soraia Attie Calil

doi:10.1016/j.jbiotec.2018.03.010

Cited by 1 publication

(1 citation statement)

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“…For obtaining SFV-VRPs, the genes coding for structural proteins were supplied by SFV-Helper2 plasmid (Berglund et al 1993). The SFV vectors used were previously obtained: SFV-RVGP and SFV-Helper2 (Benmaamar et al 2009); SFV-GFP (Puglia et al 2013) and SFV-NS3p (Lemos et al 2018). Briefly, the helper plasmid (pSFV-Helper2) and expression plasmids (pSFV-GFP, pSFV-NS3p, and pSFV-RVGP) were linearized using SpeI (for the helper plasmid) and NruI (for the expression plasmids) restriction enzymes and were confirmed by restriction digestion analysis.…”

Section: Generation Of Sfv-vrpsmentioning

confidence: 99%

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

et al. 2019

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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. Keywords BHK-21 cells Á Hepatitis C virus nonstructural protein 3 protease domain Á Rabies virus glycoprotein Á Serum-free medium Á Semliki Forest Virus Á Viral replicon particles Abbreviations SFV-VRPs Viral replicon particles of Semliki Forest Virus BHK/FBS BHK-21 cells grown in DMEM supplemented with 10% of fetal bovine serum Carlos Augusto Pereira-Died October 27th, 2017.

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