High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor

Wang, Lulan; Hu, Hongxing; Yang, Jianjun; Wang, Zhen; Kaisermayer, Christian; Zhou, Paul

doi:10.1007/s12033-011-9484-5

Cited by 35 publications

(33 citation statements)

References 42 publications

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“…3A and B). In our study with other S2 clones, we found that using Wave bioreactor stably transfected S2 clones in perfusion culture could grow to 10 8 cells per ml without compromising cell viability (53). Therefore, there is still room to improve HIV-1 VLP production by our stably transfected S2 clones.…”

Section: Discussionmentioning

confidence: 68%

“…To test the antigenicity of HIV-1 envelope glycoproteins produced by stably transfected S2 clones, we performed three sets of experiments with a panel of broadly neutralizing antibodies-2G12, b12, VRC01, PG16, and 4E10 (6, 7, 52, 55, 60, 63)-as well as the control antibody 100F4 antibody (53). The antige- nicity results of HIV-1 envelope glycoproteins are summarized in Table 2.…”

Section: Resultsmentioning

confidence: 99%

“…D7324) were purchased from Advanced Bioscience Laboratory, Inc. (Silver Spring, MD), and Aalto BioReagents (Dublin, Ireland), respectively. Human anti-gp120 antibody VRC01 and human anti-H5 hemagglutinin (HA) antibody 100F4 were produced and purified in our laboratory by using stably transfected S2 clones as described previously (53). Allophycocyanin (APC)-conjugated anti-CD8, PerCP-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated antigamma interferon (anti-IFN-␥), phycoerythrin (PE)-Cy7-conjugated anti-tumor necrosis factor alpha (anti-TNF-␣), and purified anti-CD49d and anti-CD28 antibodies were purchased from BD Bioscience (Mountain View, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Wave bioreactor 20/50EHT systems with a Wavepod process control unit (GE Healthcare) were used to grow S2 clones in fed-batch culture for HIV-1 VLP and soluble gp120 production as described before (53). Briefly, 6 ϫ 10 8 cells of the S2 clones in 300 ml of complete Express Five SFM medium were inoculated into a 2-liter cell bag and cultured at 28°C without CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

Yang

Song

et al. 2012

J Virol

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The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISAbinding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1.

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Section: Discussionmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

Yang

Song

et al. 2012

J Virol

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“…The D265A mutant was generated using specific primers and the QuikChange site-directed mutagenesis kit II (Agilent Technologies) and validated by direct sequencing. The expression vectors containing genes encoding the chimeric 100F4 or 65C6 heavy chains along with the expression vectors containing genes encoding the 100F4 or 65C6 light chain, respectively, were cotransfected into Drosophila S2 cells, and stably transfected S2 cell clones were selected as described before (47). The cAbs 65C6/IgG2a, 65C6/D265A, 100F4/IgG2a, and 100F4/D265A were produced by the stably transfected Drosophila S2 clones and purified by affinity chromatography using protein A agarose (Pierce; Thermo Fisher Scientific) and quantified by bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

Wang

Ren

Jiang

et al. 2017

J Virol

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Recent studies have shown that Fc-Fc␥ receptor (Fc␥R) interactions are required for in vivo protection against influenza viruses by broadly reactive antihemagglutinin (HA) stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single-challenge virus strains, it remains unknown whether Fc-Fc␥R interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS and whether the requirement is virus strain specific or epitope specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating Fc␥Rs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind Fc␥R. Virus neutralizing activity, binding, antibody-dependent cellular cytotoxicity (ADCC), and in vivo protection of these Abs were compared using three H5 strains, A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-Fc␥R interactions are required for in vivo protection by 100F4, but not by 65C6, and therefore, protection is not virus strain specific but epitope specific.IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-Fc␥R interactions are required for in vivo protection by broadly neutralizing antistem, but not by virus strain-specific, anti-receptor binding site (RBS), Abs. Whether such interactions are necessary for protection by Abs that recognize epitopes outside RBS is not fully understood. In the present study, we investigated in vivo protection mechanisms against three H5 strains by two pan-H5 Abs, 65C6 and 100F4. We show that although these two Abs have similar neutralizing, binding, and ADCC activities against all three H5 strains in vitro, they have divergent requirements for Fc-Fc␥R interactions to protect against the three H5 strains in vivo. The FcFc␥R interactions are required for in vivo protection by 100F4, but not by 65C6. Thus, we conclude that Fc-Fc␥R interactions for in vivo protection by pan-H5 Abs is not strain specific, but epitope specific.KEYWORDS influenza viruses, in vivo protection, monoclonal antibodies

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Advances in the Application of Perfusion Technologies toDrosophilaS2 Insects Cell Culture

Poulsen

Jongh

2014

Continuous Processing in Pharmaceutical Manufacturing

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High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor

Cited by 35 publications

References 42 publications

HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

Advances in the Application of Perfusion Technologies toDrosophilaS2 Insects Cell Culture

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