A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Published: September 29, 2023

Abstract

Source: Arevalo, M. T., et al.  Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).

This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors — encoding different viral structural proteins — in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

Protocol

1. Mammalian Expression System for Generation of Influenza H3N2 VLP

  1. Subclone viral structural glycoproteins hemagglutinin (HA), neuraminidase (NA), and human immunodeficiency virus (HIV) Gag (group-specific antigen) p55 into eukaryotic expression vectors, such as previously described pTR600.
  2. Amplify DNA in chemically competent Escherichia coli (e.g., DH5α) and isolate transfection-grade plasmid using a plasmid purification kit per manufacturer's instructions. The amount of DNA is dependent on yield and the user's needs.
  3. Maintain mammalian cell line, 293T, in growth media containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO2 in a humidified incubator.
  4. Grow cells such that a T-150 tissue culture flask contains 45-75 x 106 cells per flask such that the flask obtains complete cell adherence and 85-95% cell confluency by the following day. Inspect cells under the microscope for the desired confluency.
    Note: High cell density is typically recommended to yield optimal protein expression with minimal cytotoxicity when using commercial transfection reagents however over-confluency may result in accumulation of cellular waste by-products and reduce cell viability.
  5. On the day of transfection, prepare liposome and DNA mixture as per the manufacturer's recommendations and transfect DNA cells with a DNA composition of 1:1:2 of HA:NA:Gag with a total DNA quantity of 40 µg per T-150 flask. Dilute DNA and liposome solutions in serum-free transfection media without antibiotics such that each flask contains a total volume of 20-30 ml.
    Note: The ratio of gene constructs may require user optimization. Although not demonstrated here, a similar transfection procedure has been performed with respiratory syncytial virus (RSV) F and HIV Gag at a 1:1 ratio. For DENV and CHIKV single-expression plasmids, a total of 20 µg of DNA per T-150 flask is used for optimal expression. DNA:transfection reagent ratios are user-optimized. Use recommended transfection medium based on transfection method, i.e., polycationic lipid transfections recommend reduction or absence of fetal bovine serum thus media may be supplemented with growth hormones and trace elements to support growth in the absence of serum.
  6. Return flasks to 37 °C with 5% CO2 incubator. For a volume of 200 ml, use 9 T-150 flasks. The cells are maintained in a transfection culture medium until the day of VLP harvest.
  7. Harvest culture supernatant from cells after 72-96 hr post-transfection depending on antigen, or when cell viability has decreased to 70-80%, as estimated by microscopic inspection. Transfer supernatant into 50 ml conical tubes and spin the cells down at 500 × g for 5 min at 4 °C to pellet cellular debris.
    Note: Replacement of 3-day supernatant with fresh pre-warmed, serum-free transfection media to adherent cells will yield a second lot of VLPs with similar or slightly reduced yield and should be optimized by the user.
  8. Pool supernatant and filter through a 0.22 µm pore membrane before sedimentation via ultracentrifugation.

Declarações

The authors have nothing to disclose.

Materials

Plasmid Maxi Kit Qiagen 12163
DMEM Cellgro 10-013
Lipofectamine Life Technologies L3000075 Lipofectamine 2000, Lipofectamine 3000
Opti-MEM I Life Technologies 31985062
Cimarec I 6 multipoint stirrer plate ThermoFisher Scientific 50094596
Polyclear ultracentrifuge tubes Seton 7025
0.22 μm vacuum filter top (500 mL) Nalgene 569-0020
Glycerol Sigma G5516

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A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System. J. Vis. Exp. (Pending Publication), e21700, doi: (2023).

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