An Alternative Perfusion Approach for the Intensification of Virus-Like Particle Production in HEK293 Cultures

Lavado‐García, Jesús; Cervera, Laura; Gòdia, Francesc

doi:10.3389/fbioe.2020.00617

Cited by 20 publications

(23 citation statements)

References 51 publications

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“…On the other hand, we investigated if the fusion protein GH-RVG was present in the structure of the observed particles, anchored in the VLPs envelope as is described when a membrane glycoprotein is co-expressed with Gag protein [ 37 , 38 , 39 , 40 ]. Therfore, we performed immunogold labelling of the purified cVLPs and analyzed it by TEM ( Figure 5 C,D).…”

Section: Resultsmentioning

confidence: 99%

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus

et al. 2021

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Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals.

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Section: Resultsmentioning

confidence: 99%

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus

et al. 2021

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“…[ 18 ] In terms of specific Gag VLP productivity, stable High Five cell pools achieved 0.5 ng 10 −6 cell·day, resulting in comparable levels to those obtained in clonal High Five cell lines [ 19 ] and 1.8‐fold higher in comparison to clonal Sf9 cell lines. [ 18 ] The VLP production yields shown in this work are still lower than those obtained with the BEVS, [ 22 ] but the development of new strategies for the intensified production of recombinant proteins [ 40 ] as well as the specific supplementation of cell cultures [ 41 ] open a window of opportunity to further increase VLP yields.…”

Section: Resultsmentioning

confidence: 99%

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

Puente‐Massaguer

Grau-Garcia

Strobl

et al. 2020

Biotechnology Journal

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Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

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“…The perfusion approach enables continuous addition of fresh medium and removal of by-products, so that high cell concentration can be achieved. Thus, it has been used for production of recombinant therapeutic proteins (CHO cells), virus-like particles (HEK 293 cells), and vaccines (avian cells) [7] , [8] , [19] .…”

Section: Discussionmentioning

confidence: 99%

The efficient development of a novel recombinant adenovirus zoster vaccine perfusion production process

Nie¹,

Sun²,

Feng³

et al. 2022

Vaccine

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An Alternative Perfusion Approach for the Intensification of Virus-Like Particle Production in HEK293 Cultures

Cited by 20 publications

References 51 publications

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

The efficient development of a novel recombinant adenovirus zoster vaccine perfusion production process

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