Expression of full-length HER2 protein in Sf 9 insect cells and its presentation on the surface of budded virus-like particles

Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag‐based virus‐like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo‐TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion‐derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23‐fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag‐eGFP polyprotein were obtained in High Five cells (3.6‐fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7‐ and 1.5‐fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five‐derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry

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“…virus (HIV) is conventionally the preferred scaffold for protein display in the lipid envelope (5,6).…”

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confidence: 99%

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

et al. 2020

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“…Yeast [1] [2], mammalian [3] [4], insect [5] [6] and bacterial cells [7][8] [9] have been used to display recombinant proteins on their cell surfaces for various cell-surface display applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes [10] [11]. Among bacterial host strains, Escherichia coli is the most widely used as it is genetically well-characterised and has extraordinary versatility due to its rapid growth and ease of genetic manipulation [12].…”

Section: Introductionmentioning

confidence: 99%

Decorating the Surface of Escherichia Coli with Bacterial Lipoproteins: A Comparative Analysis of Different Display Systems

Nicchi¹,

Giuliani²,

Giusti³

et al. 2020

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Background: The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins.Results: In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37°C and 25°C as demonstrated by FACS analysis, confocal and transmission electron microscopy.Conclusions: To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, , AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

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“…To address this limitation, we applied SweetBac technology [22] to generate mammalian-like N -glycan structures on the antigen. Overall, our VLP technology allowed us to (I) provide HER2 in an authentic conformation and as a complete molecule [23] and to (II) investigate the impact of different glycosylation patterns on the immunogenic effect of HER2-displaying VLPs.…”

Section: Introductionmentioning

confidence: 99%

A HER2-Displaying Virus-Like Particle Vaccine Protects from Challenge with Mammary Carcinoma Cells in a Mouse Model

et al. 2019

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Human epidermal growth factor receptor-2 (HER2) is upregulated in 20% to 30% of breast cancers and is a marker of a poor outcome. Due to the development of resistance to passive immunotherapy with Trastuzumab, active anti-HER2 vaccination strategies that could potentially trigger durable tumor-specific immune responses have become an attractive research area. Recently, we have shown that budded virus-like particles (VLPs) produced in Sf9 insect cells are an ideal platform for the expression of complex membrane proteins. To assess the efficacy of antigen-displaying VLPs as active cancer vaccines, BALB/c mice were immunized with insect cell glycosylated and mammalian-like glycosylated HER2-displaying VLPs in combination with two different adjuvants and were challenged with HER2-positive tumors. Higher HER2-specific antibody titers and effector functions were induced in mice vaccinated with insect cell glycosylated HER2 VLPs compared to mammalian-like glycosylated counterparts. Moreover, insect cell glycosylated HER2 VLPs elicited a protective effect in mice grafted with HER2-positive mammary carcinoma cells. Interestingly, no protection was observed in mice that were adjuvanted with Poly (I:C). Here, we show that antigen-displaying VLPs produced in Sf9 insect cells were able to induce robust and durable immune responses in vivo and have the potential to be utilized as active cancer vaccines.

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Expression of full-length HER2 protein in Sf 9 insect cells and its presentation on the surface of budded virus-like particles

Cited by 19 publications

References 43 publications

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

Decorating the Surface of Escherichia Coli with Bacterial Lipoproteins: A Comparative Analysis of Different Display Systems

A HER2-Displaying Virus-Like Particle Vaccine Protects from Challenge with Mammary Carcinoma Cells in a Mouse Model

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