Identifying parameters to improve the reproducibility of transient gene expression in High Five cells

Bleckmann, Maren; Schürig, Margitta; Endres, Michelle; Samuels, Anke; Gebauer, Daniela; Konisch, Nadine; Heuvel, Joop van den

doi:10.1371/journal.pone.0217878

Cited by 20 publications

(25 citation statements)

References 52 publications

(57 reference statements)

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“…SARS-CoV-2 RBD-SD1 (aa319-591) according to Wrapp et al 2020 33 , S1 subunit (aa14-694), S1-S2 (aa14-1210, with proline substitutions at position 986 and 987 and "GSAS" substitution at the furin site) and extracellular ACE2 were produced in insect cells using a plasmid based baculovirus free system 34 as well as in Expi293F cells. All antigens with exception of S1-S2 were produced with human IgG1 Fc part, murine IgG2a Fc part or with 6xHis tag in both expression systems.…”

Section: Sars Cov2 Spike Domains or Subunits And Human Ace2 Were Prodmentioning

confidence: 99%

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Bertoglio

Meier

Langreder

et al. 2020

Preprint

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COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein.The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronavirus has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti SARS2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. Furthermore, these antibodies neutralized active SARS-Cov-2 virus infection of VeroE6 cells. All 17 were all able to bind the isolated RBD, four of them with sub-nanomolar EC50. Epitope analysis of the antibodies revealed that six bind at the RBD-ACE2 interface and two on the opposite side of the domain. Universal libraries from healthy donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation. 4/34 Main textIn 2015 Menachery et al. wrote: "Our work suggests a potential risk of SARS-CoV reemergence from viruses currently circulating in bat populations." 1 . Four years later, a novel coronavirus causing a severe pneumonia was discovered and later named SARS-CoV-2. The outbreak started on a sea food market in Wuhan, Hubei province (China) at the end of 2019. The disease was named COVID-19 (coronavirus disease 2019) by the World Health Organization (WHO). Sequencing showed high identity to bat corona viruses (CoV, in particular RaTG13), beta-CoV virus causing human diseases like SARS and MERS and, to a lesser extent, the seasonal CoV hCoV-OC43 and HCov-HKU1 2,3 . The spike (S) protein of SARS-CoV-2, as well as SARS-CoV, binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) which is expressed on lung cells, heart, kidney and intestine cells and acts as receptor for virus entry. S protein consists of the N-terminal S1 subunit, which includes the receptor binding domain (RBD), and the Cterminal S2 subunit which is anchored to the viral membrane and is required for trimerization and fusion of the virus and host membrane 4-6 . The membrane bound host protease TMPRSS2 is responsible for S protein priming by cleavage of specific sites between S1 and S2. In addition to proteolytic activation of the S2' site, conformational changes and viral entry 7-10 .Antibodies against the...

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Section: Sars Cov2 Spike Domains or Subunits And Human Ace2 Were Prodmentioning

confidence: 99%

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Bertoglio

Meier

Langreder

et al. 2020

Preprint

Self Cite

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“…Therefore, the expression of the GOI is lost over time after cell division. In recent years, several studies have shown that suspension-adapted High Five and Sf9 cells are ideal hosts for the production of reporter proteins [15][16][17], antibodies [18][19][20] and surface proteins [21] in this BV-free environment. Still, the assessment of the insect cell/TGE system to produce more complex products such as VLPs remains to be investigated.…”

Section: Introductionmentioning

confidence: 99%

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

et al. 2020

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High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

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“…Supplementation of the media. Different additives (Pluronic F68, DMSO), known to have an impact on transfection 7 , and different media, known to be compatible with this method 25 (EX-CELL405 (Merck) and Sf-900™ III SFM (ThermoFisher)), were tested for their impact on expression ( Fig. 3a).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, the plasmid-based production in insect cells without the use of baculovirus was reported [21][22][23][24][25][26] . Different protocols and expression vectors exist but in each case the expression vector is quite efficiently delivered by Polyethylenimine (PEI) and no baculovirus is required.…”

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confidence: 99%

Baculovirus-free insect cell expression system for high yield antibody and antigen production

Korn

Schäckermann

Kirmann

et al. 2020

Sci Rep

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Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.

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Identifying parameters to improve the reproducibility of transient gene expression in High Five cells

Cited by 20 publications

References 52 publications

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

Baculovirus-free insect cell expression system for high yield antibody and antigen production

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