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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.

RESUMEN

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.

RESUMEN

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.

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The original PDF version of this Article contained errors in the spelling of Luiz Carlos Caires-Júnior, Uirá Souto Melo, Bruno Henrique Silva Araujo, Alessandra Soares-Schanoski, Murilo Sena Amaral, Kayque Alves Telles-Silva, Vanessa van der Linden, Helio van der Linden, João Ricardo Mendes de Oliveira, Nivia Maria Rodrigues Arrais, Joanna Goes Castro Meira, Ana Jovina Barreto Bispo, Esper Abrão Cavalheiro, and Robert Andreata-Santos, which were incorrectly given as Luiz Carlos de Caires Jr., UiráSouto Melo, Bruno Silva Henrique Araujo, Alessandra Soares Schanoski, MuriloSena Amaral, Kayque Telles Alves Silva, Vanessa Van der Linden, Helio Van der Linden, João Mendes Ricardo de Oliveira, Nivia Rodrigues Maria Arrais, Joanna Castro Goes Meira, Ana JovinaBarreto Bispo, EsperAbrão Cavalheiro, and Robert Andreata Santos. Furthermore, in both the PDF and HTML versions of the Article, the top panel of Fig. 3e was incorrectly labeled '10608-1' and should have been '10608-4', and financial support from CAPES and DECIT-MS was inadvertently omitted from the Acknowledgements section. These errors have now been corrected in both the PDF and HTML versions of the Article.

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Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

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Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4x10(4) SFV-NS3p/mu L and 4.0x10(2) HCVpp-NS3p/mu L. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.

RESUMEN

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

RESUMEN

Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4x10(4) SFV-NS3p/mu L and 4.0x10(2) HCVpp-NS3p/mu L. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.

RESUMEN

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.

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The expression of recombinant viral envelope glycoproteins in S2 (Drosophila melanogaster) has been performed with good results. This chapter contains protocols for the utilization of this system for the expression and analysis of proteins presented in cell plasma membrane.

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The transient transfection process has been developed to allow rapid production of recombinant proteins. In this paper, we describe the transient expression of recombinant rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells. Different cell transfection reagents were evaluated, together with the effects of different cell cultivation procedures on RVGP expression. Yields of RVGP in the range 50-90ng/10(7) cells were obtained in multi-well plate transfection experiments, where it was observed that RVGP expression was linked to the DNA concentration. RVGP expression was 1.3 times higher using 10µg rather than 5µg of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15µg decreasing RVGP expression 1.5-fold for cells transfected with polyethylenimine (PEI) and 1.6-fold for cells transfected with cationic lipid. The results of shake flask transfection indicated that S2 cells were more effectively transfected in suspension than under static conditions. RVGP yields of 182.2ng/10(7) cells (PEI), 201ng/10(7) cells (calcium phosphate), and 215ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309ng/mL) was found for cells transfected with cationic lipid. This value was 1.21 and 1.16 times higher, respectively, than for cells transfected with PEI (253.4ng/mL) and calcium phosphate (237.2ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138ng/10(7) cells for transfection using PEI and calcium phosphate, respectively. A comparison of the different transfection reagents (calcium phosphate, cationic lipid, and cationic polymer) showed no significant differences in RVGP expression when shake flasks were used. Overall, the data indicated that transient expression in D. melanogaster S2 cells is a practical way of synthesizing RVGP for use in structural and functional studies.

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