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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

Expression and Purification of Virus-like Particles for Vaccination

Published: June 2nd, 2016

DOI:

10.3791/54041

1Center for Vaccines and Immunology, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user's microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Virus-like particles (VLPs) are an approved technology for human vaccination. In fact, some of the more contemporarily licensed vaccines, including the human papillomavirus (HPV) and hepatitis Β (HepΒ) vaccines employ this approach. VLPs are formed from structural proteins capable of self-assembly. The assembled particles mimic viral morphologies, but cannot infect or replicate because they lack viral genomes. VLPs can be expressed and purified from a number of prokaryotic and eukaryotic systems. A review of the literature revealed that different expression systems are employed at the following rates: bacteria - 28%, yeast - 20%, plant - 9%, insect - 28....

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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 p55 into eukaryotic expression vectors, such as previously described pTR600.7
  2. Amplify DNA in chemically competent Escherichia coli (e.g., DH5α) and isolate transfection-grade plasmid using a plasmid purification kit as per manufacturer's instructions. Amount of DNA is dependent on yi.......

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VLP yields were variable by viral antigen construct design. In this protocol, we have demonstrated use of insect and mammalian cells for production of SVP or VLPs in supernatant and purification by ultracentrifugation. Four subtypes of DENV prM/E structural gene expression cassettes were used to construct the versions of DENV SVPs (demarcated as 1-4) in Table 1 and demonstrate a range between 1.1-2.6 mg of total protein in 0.6 ml volumes. For VLPs that require a three gen.......

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We have used the techniques described above to successfully express and purify SVPs and VLPs composed of multiple structural proteins for various pathogens. In general, we use mammalian expression systems to generate our VLPs. However, in our hands, mammalian-cell derived CHIK VLP yields were low. CHIK VLP yield was more robust when using A recombinant baculovirus-insect cell system. In general, baculovirus-insect cell systems yield higher amounts of recombinant proteins, which may result from the higher cell densities t.......

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We wish to acknowledge the prior members of the Ross lab who have helped optimize the protocol for maximal efficiency and yields.

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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
Bac-to-Bac Baculovirus Expression System Life Technologies 10359-016
Cimarec I 6 multipoint stirrer plate ThermoFisher Scientific 50094596
Polyclear ultracentrifuge tubes Seton 7025 Confirm appropriate tubes for ultracentrifuge and bucket size
Micro BCA Protein Assay Kit ThermoFisher Scientific 23235
Phosphate citrate buffer Sigma P4922
o-phenylenediamine dihydrochloride  Sigma P8287
0.22 μm vacuum filter top (500 mL) Nalgene 569-0020
Glycerol Sigma G5516
H2SO4 Sigma 339741 
Sf-900 II SFM ThermoFisher Scientific 10902-096

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