Expression and Purification of Virus-like Particles for Vaccination

Summary

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.

Abstract

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.

Introduction

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%, and mammalian - 15%¹. Of note, HPV vaccines based on L1 capsid protein are produced in yeast (Gardasil) or in an insect cell system (Cervarix)². HepΒ vaccines, Recombivax and Engerix-Β, are also produced in yeast, and are composed of HepΒ surface antigen^3,4.

We use mammalian and insect cell expression systems to produce VLPs requiring co-expression of multiple structural proteins for assembly. Our work focuses on designing, producing, and purifying VLP-based vaccines against human pathogens: influenza virus, respiratory syncytial virus (RSV), dengue virus (DENV), and chikungunya virus (CHIKV). Our methods are flexible enough to allow for co-expression of the multiple structural proteins from multiple expression plasmids, or a single expression plasmid (Figure 1). Previously, we produced and purified H5N1 VLPs assembled from the co-expression of plasmids encoding influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M1) in human embryonic kidney 293T cells^5,6. The genes were codon-optimized for expression in mammalian cells and cloned into pTR600, a eukaryotic expression vector containing the cytomegalovirus immediate-early promoter plus intron A for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation signal for termination of transcription⁷. A similar approach using the three-plasmid co-expression of HA, NA (Figure 1A) and an alternative viral matrix protein, HIV Gag p55, was used for generation of human seasonal influenza subtype H3N2 VLPs in this study and has been shown to generate VLPs of similar size as influenza particles⁸. Although influenza vaccines predominantly elicit anti-HA antibodies, the addition of influenza neuraminidase mediates sialidase activity to enable VLP budding from transfected cells⁹ and also present additional immunogenic targets. To produce RSV VLPs, we also selected the unrelated core of HIV Gag to design prototypical vaccines that present exclusively RSV surface glycoproteins to further demonstrate flexibility of VLP formation using HIV Gag, as previously described and characterized by electron microscopy^6,10. Others have previously shown that VLPs presenting RSV glycoproteins can be assembled using various viral components from Newcastle Disease virus (NDV)¹¹, and influenza matrix¹². Full-length surface glycoprotein sequences were utilized in this study to retain native conformations that may be necessary for functional receptor binding and antibody recognition assay through enzyme-linked immunosorbent assay (ELISA).

Our examples for use of single plasmid expression systems to generate particles are DENV and CHIKV. In the case of DENV, we can produce subviral particles (SVPs) with no capsid in 293T cells from a single plasmid containing a prM/E structural gene expression cassette¹³. The term SVP is used to denote the lack of a core or capsid protein in the assembly of viral structural proteins. CHIK VLPs can also be produced using a single plasmid containing a CHIKV structural gene cassette, encoding capsid and envelope proteins, or in insect cells by infecting with a recombinant baculovirus encoding the same structural gene cassette optimized for insect cell expression (Figure 1B-C).

The end result of the expression approaches discussed above is the release of VLPs into cell culture medium that can then be purified via ultracentrifugation through a 20% glycerol cushion. In this report, we present methods to express and purify these VLPs from mammalian and insect cell systems.

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 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 yield and 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 flask obtains complete cell adherence and 85-95% cell confluency by the following day. Inspect cells under the microscope for 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: 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 are 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 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 user.
8. Pool supernatant and filter through a 0.22 µm pore membrane before sedimentation via ultracentrifugation.
9. Test the hemagglutination activity of the HA-expressing VLPs by standard hemagglutination assay¹⁴ using 0.8% turkey or mammalian red blood cells or proceed with antigen-specific ELISA. See Figure 2 for representative results.

2. CHIK VLP Expression Using Baculovirus/Insect Cell System

1. Generate recombinant baculovirus expressing chikungunya virus capsid and envelope proteins (C-E3-E2-6K-E1) from S-27 strain using a commercial baculovirus expression system.
2. Culture Spodoptera frugiperda Sf9 cells in serum-free Sf9 growth medium with 100 U/ml penicillin and 100 µg/ml streptomycin at 28 °C. Use 3-5 x 10⁵ c/ml to initiate spinner flask cultures for suspension cell growth. Passage routinely when they reach cell densities of 2-4 x 10⁶ c/ml (every 3-4 days).
3. Culture Sf9 cells in suspension in spinner flasks with stirring at 130 rpm on a multipoint stirrer plate system. For proper aeration, maintain culture volumes at no more than half the volume of the spinner flask.
4. For expression of CHIK VLPs, infect Sf9 cells at a density of 2 x 10⁶ cells/ml with recombinant baculovirus at a multiplicity of infection of 1 and return to 28 °C incubator. Typically, infect one or two spinner flasks, containing 250 ml Sf9 cells.
   Note: While we do not use this method, Sf9 cells may be cultured in shaker flasks at 28 °C using a shaker platform.
5. Harvest cultures after 72-96 hr post-infection, or when cell viability has decreased to 70-80% as determined by Trypan Blue Exclusion according to manufacturer's protocol.
   Note: The cells continue to proliferate while infected and there is a ~80% viability in Sf9 cultures infected with recombinant CHIK VLP baculovirus at 3 days post-infection (dpi). Baculovirus-infected cells are larger in appearance. Morphology may also change from round to oblong. In late-stages of baculovirus infections, the cells began to lyse.
6. Transfer cultures directly from suspension culture into 50 ml conical tubes and spin the cells down at 500 x g for 5 min at 4 °C.
7. Collect supernatants and filter through a 0.22 µm pore membrane before sedimentation via ultracentrifugation.

3. Sedimentation/Purification of VLP/SVPs

1. Sterilize 25 mm x 89 mm open-top ultracentrifuge tubes with 70% ethanol in biosafety hood. Ensure the ethanol has dried off before use.
2. Load up to 32 ml of supernatant into a clean tube. A minimal volume of 25 ml is recommended to prevent collapse and damage of inadequately filled ultracentrifuge tubes.
3. Carefully underlay supernatants with sterile 3 ml 20% glycerol in PBS (v/v). Make sure tubes are balanced.
4. Spin at 135,000 x g for 4 hr at 4 °C.
5. Aspirate supernatant, ensuring the pellet does not dislodge from tube.
6. Resuspend sedimented VLPs at bottom of the tubes with sterile PBS by vigorously pipetting up and down. The amount of PBS needed to resuspend VLPs depends on VLP total protein yield and downstream applications. Typically, resuspend each VLP pellet in at least 100 µl.
7. Store samples 4 °C for short-term storage and -80 °C storage for long-term storage.

4. Determining Protein Yields and Specific Antigen Yields

1. Determine the total protein content using a commercial protein quantification method, such as BCA assay as per manufacturer's protocol.
2. To assess specific antigen content, perform direct enzyme-linked immunosorbent assay (ELISA) by coating serial dilutions of standard antigen and 2-5 µg of total protein sample on to an ELISA 96-well flat bottom plate.
   1. Follow with antigen-specific antibodies to probe the presence of the antigens on the plate and use conventional ELISA development substrates to produce detectable absorbance for microplate reader. See Figure 2 for representative results of HA and ELISA assay for a sample HA-expressing VLP; many combinations of HA and NA are possibly but yields may differ upon HA-NA compatibility¹⁵.
      Note: Antigen-specific antibodies have variable affinities and it is recommended that endpoint-dilution titration of antibodies be determined before performing VLP quantification. Commercial antibodies will have suggested concentration or dilution ranges for use in ELISA, as well as suggested protocols.

Results

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...

Discussion

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...

Materials

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