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

Virus-like particles, or VLPs, mimic the virus structure but lack viral genetic material, rendering them non-virulent. VLPs possess surface-bound antigenic epitopes recognized by immune cells, making them ideal vaccine candidates.

To generate recombinant influenza VLPs, take eukaryotic expression vectors.

Two vectors contain genes coding for hemagglutinin, or HA, and neuraminidase, or NA — structural proteins of the influenza virus. The third vector contains the gag gene, encoding core proteins of the human immunodeficiency virus.

Add a cationic lipid-based transfection reagent. The positively charged lipid head group interacts with negatively charged DNA, forming a bilayer around the vectors — generating liposome transfection complexes.

Add complexes onto cultured mammalian host cells suitable for VLP production, and incubate. Transfection complexes enter cells through endocytosis — the liposome membrane fuses with the endosome membrane to release the vectors into the cytoplasm.

Vectors enter the nucleus, leading to the expression of the viral genes. Upon HA and NA synthesis on the endoplasmic reticulum, the proteins translocate to the plasma membrane via the secretory pathway.

The core proteins are synthesized in the cytoplasm and transported to the assembly site to anchor at the plasma membrane. Accumulated viral components undergo self-assembly into VLPs and bud from the host cell.

Harvest the released VLPs for downstream processing.

On the day of transfection, prepare liposome and DNA solutions per the manufacturer's recommendations. Transfect the DNA cells with a DNA composition of 1:1:2 of HA:NA:Gag and a total DNA quantity of 40 micrograms per T-150 flask. Repeat this step to create nine T-150 flasks for a total volume of 200 milliliters.

Next, dilute the DNA and liposome solutions in serum-free transfection media without antibiotics so that each flask contains a total volume of 24 milliliters. Return the flasks to the incubator and maintain the cells in transfection culture medium until the day of virus-like particle or VLP, harvest.

Transfer the supernatant into 15-milliliter conical tubes to harvest the culture from the cells after 72 to 96 hours post-transfection. Spin the cells down to pellet the cellular debris. Collect the supernatants and filter them through a 0.22-micron pore membrane.