A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System
JoVE Encyclopedia of Experiments
Immunology
0 views • 3:09 min • July 8th, 2025
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.
Overview
This article discusses the production of virus-like particles (VLPs) as potential vaccine candidates. VLPs mimic the structure of viruses but do not contain viral genetic material, making them non-virulent and safe for use in immunization.
Key Study Components
Area of Science
- Vaccine development
- Virology
- Immunology
Background
- Virus-like particles are effective in eliciting immune responses.
- They can be produced using recombinant DNA technology.
- Influenza VLPs can be generated using specific eukaryotic expression vectors.
- Understanding the assembly and release of VLPs is crucial for vaccine efficacy.
Purpose of Study
- To outline a method for generating recombinant influenza VLPs.
- To explore the use of cationic lipid-based transfection reagents in VLP production.
- To provide a detailed protocol for transfecting mammalian cells with VLP components.
Methods Used
- Utilization of eukaryotic expression vectors containing HA, NA, and gag genes.
- Application of cationic lipid-based transfection reagents to facilitate DNA uptake.
- Incubation of transfected cells to allow for VLP assembly.
- Harvesting and filtering of VLPs from cell culture supernatants.
Main Results
- Successful transfection of mammalian cells led to VLP production.
- VLPs were harvested after 72 to 96 hours post-transfection.
- Filtration through a 0.22-micron membrane ensured removal of cellular debris.
- VLPs exhibited structural characteristics similar to native viruses.
Conclusions
- Recombinant influenza VLPs can be efficiently produced using the outlined method.
- The use of lipid-based transfection reagents is effective for VLP generation.
- This approach holds promise for developing safe and effective vaccines.
Frequently Asked Questions
Executive Industry Relevance
The generation of influenza virus-like particles (VLPs) in mammalian systems enables the production of structurally authentic, non-infectious viral mimics for vaccine research and immunogenicity studies. This approach supports early-stage vaccine candidate evaluation and de-risks downstream translational efforts by providing scalable, reproducible antigen sources. The method is strategically positioned for rapid response to emerging influenza strains and portfolio diversification in vaccine R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of antigenic structure and immune recognition in a controlled system.
- Supports biological de-risking by providing non-virulent, structurally relevant particles for immunogenicity assessment.
- Facilitates predictive confidence in antigen selection for vaccine development pipelines.
Screening & Assay Development
- Provides validated VLPs for use in immunoassays and neutralization screens.
- Enables assay standardization and reproducibility through consistent VLP production.
- Supports scalable preparation of antigenic material for high-throughput screening platforms.
Translational & Preclinical Research
- Aligns with disease-relevant antigen presentation for preclinical immunogenicity studies.
- Ensures continuity from discovery to preclinical validation by supplying authentic viral mimics.
- Reduces risk in translational advancement by enabling early immunological profiling.
Pipeline & Workflow Integration
This VLP generation technique integrates at the interface of antigen discovery, assay development, and preclinical immunogenicity evaluation within the vaccine R&D continuum.
- Discovery Biology: Supports hypothesis testing of antigenicity and immune response potential.
- Screening: Delivers reproducible, quantitative VLP outputs for assay readiness.
- Analytics: Enables measurement of immune recognition and comparative antigenicity across constructs.
- Translational Research: Provides a bridge to preclinical vaccine candidate assessment with structurally relevant particles.
- Enterprise Reuse: Establishes a platform for rapid adaptation to new influenza strains or other viral targets.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in antigen selection and reduces mechanistic ambiguity in immune response studies.
- Operational Value: Standardizes VLP production for reproducibility and scalability across R&D teams.
- Strategic Value: Accelerates go/no-go decisions and enhances capital efficiency in vaccine portfolio management.
- Portfolio Impact: Enables risk-adjusted prioritization of vaccine candidates and supports rapid response to emerging threats.
Implementation Considerations
- Requires expertise in mammalian cell culture and recombinant DNA transfection.
- Demands access to cell culture infrastructure and analytical tools for VLP characterization.
- Necessitates cross-team standardization of transfection and harvest protocols for reproducibility.
- May require adaptation for different viral targets or host cell systems.
- Dependent on optimization of DNA ratios and transfection conditions for maximal VLP yield.
Biopharma FAQs
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Last updated: 22 August 2026