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

Protocolo

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.

Materiales

Name	Company	Catalog Number	Comments
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