A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Podsumowanie

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Streszczenie

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a "Plug-and-Display" vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

Wprowadzenie

As a potential vaccine platform, outer membrane vesicles (OMVs) have attracted more and more attention in recent years^1,2. OMVs, mainly secreted naturally by gram-negative bacteria³, are spherical nanoscale particles composed of a lipid bilayer, usually in the size of 20-300 nm⁴. OMVs contain various parental bacterial components, including bacterial antigens and pathogen-associated molecular patterns (PAMPs), which serve as solid immune potentiators⁵. Benefiting from their unique components, natural vesicle structure, and great genetic engineering modification sites, OMVs have been developed for use in many biomedical fields, including bacterial vaccines⁶, adjuvants⁷, cancer immunotherapy drugs⁸, drug delivery vectors⁹, and anti-bacterial adhesives¹⁰.

The SARS-CoV-2 pandemic, which has spread worldwide since 2020, has taken a heavy toll on global society. The receptor-binding domain (RBD) in spike protein (S protein) can bind with human angiotensin-converting enzyme 2 (ACE2), which then mediates the entry of the virus into the cell^11,12,13. Thus, RBD seems to be a prime target for vaccine discovery^14,15,16. However, monomeric RBD is poorly immunogenic, and its small molecular weight makes it difficult for the immune system to recognize, so adjuvants are often required¹⁷.

In order to increase the immunogenicity of RBD, OMVs displaying polyvalent RBDs were constructed. Existing studies using OMV to display RBD usually fuse RBD with OMV to be expressed in bacteria¹⁸. However, RBD is a virus-derived protein, and prokaryotic expression is likely to affect its activity. To solve this problem, the SpyTag (ST)/SpyCatcher (SC) system, derived from Streptococcus pyogenes, was used to form a covalent isopeptide with OMV and RBD in a conventional buffer system¹⁹. The SC domain was expressed with Cytolysin A (ClyA) as a fusion protein by bioengineered Escherichia coli, and ST was expressed with RBD via the HEK293F cellular expression system. OMV-SC and RBD-ST were mixed and incubated overnight. After purification by ultracentrifugation or size-exclusion chromatography (SEC), OMV-RBD was obtained.

Protokół

1. Plasmid construction

1. Insert DNA encoding SpyCatcher sequence (Supplementary File 1) into an ampicillin-resistant pThioHisA-ClyA plasmid (see Table of Materials) between the BamH I and Sal I sites to construct the plasmid pThioHisA ClyA-SC following a previously published report²⁰.
2. Ligate the synthesized SpyTag-RBD-Histag fusion gene (Supplementary File 1) into a pcDNA3.1 plasmid (see Table of Materials) between the BamH I and EcoR I sites to construct the plasmid pcDNA3.1 RBD-ST following a previously published report¹⁹.

2. OMV-SC preparation

1. Perform ClyA-SC transformation following the steps below.
   1. Add 5 µL of pThioHisA ClyA-SC plasmid solution (50 ng/µL) to 50 µL of BL21 competent strain, gently blow, and let cool on ice for 30 min.
   2. Place the solution for 90 s in the water bath at 42 °C, and then immediately put the mixed solution on ice for 3 min.
   3. Add 500 µL of LB medium to the bacterial suspension and, after mixing, culture at 220 rpm at 37 °C for 1 h.
   4. Plate all the transformation onto an LB agar plate containing ampicillin (100 µg/mL, see Table of Materials) and culture overnight at 37 °C.
      NOTE: In subsequent experiments, the ampicillin was kept at the same concentration in the medium. If not, this may cause loss of plasmids in the bacteria.
2. For OMV-SC production, perform the steps below.
   1. Isolate a single colony from the plate (step 2.1.4.) to 20 mL of LB (ampicillin-resistant) medium and culture overnight at 220 rpm at 37 °C.
   2. Inoculate the bacterial solution (from step 2.2.1.) into 2 L medium, culture at 220 rpm, 37 °C for 5 h until the logarithmic growth stage (the OD_600nm is between 0.6 to 0.8).
   3. When the OD at 600 nm of the bacterial solution reaches 0.6-0.8, add isopropyl beta-D-thiogalactopyranoside (IPTG, see Table of Materials) to make the final concentration of the bacterial solution to be 0.5 mM, and then culture overnight at 220 rpm at 25 °C.
3. Perform OMV-SC purification.
   1. Centrifuge the bacterial solution at 7,000 x g at 4 °C for 30 min.
   2. Filter the supernatant with a 0.22 µm membrane filter, then concentrate it using a 100 kD ultrafiltration membrane or hollow fiber column (see Table of Materials).
   3. Filter the concentrate through a 0.22 µm membrane filter, then centrifuge it at 150,000 x g at 4 °C for 2 h using an ultracentrifuge (see Table of Materials), and discard the supernatant with a pipette.
   4. Resuspend the precipitation with PBS and store it at −80 °C. The solution can maintain long-term stability at −80 °C.

3. RBD-ST preparation

1. Perform RBD-ST transfection following the steps below.
   1. Select an appropriate eukaryotic expression system (e.g., HEK293F) and culture the cells overnight at 130 rpm at 37 °C after recovery.
   2. Add 20 µL of HEK293F cell solution into the automated cell counter (see Table of Materials), record the number of cells, adjust the concentration to 1 x 10⁶ cells/mL, and then culture the cells at 130 rpm at 37 °C for 4 h.
   3. Filter RBD-ST plasmid through a 0.22 µm membrane filter, and add 300 µg of plasmids into the cell culture medium (see Table of Materials) until the final volume is 10 mL; shake for 10 s.
   4. Heat PEI (1 mg/mL, see Table of Materials) to 65 °C in the water bath, mix 0.7 mL of PEI with 9.3 mL of cell culture medium, and shake intermittently for 10 s.
      NOTE: Do not shake the solution vigorously. Otherwise, the resulting bubbles may affect the transfection efficiency.
   5. Add plasmid solution to the PEI solution, shake the mixture intermittently for 10 s, and incubate it at 37 °C for 15 min.
   6. Add the mixture to 280 mL of cell culture medium and culture at 130 rpm at 37 °C for 5 days.
2. Perform RBD-ST purification.
   1. Centrifuge the cells at 6,000 x g at 25 °C for 20 min and use a pipette to collect the supernatant.
   2. Fill the column with 2 mL of Ni-NTA agarose (see Table of Materials) and wash it 3x with 3x PBS.
   3. Add imidazole (see Table of Materials) into the cell supernatant to make the final concentration of 20 mM, and load the cell supernatant 2x.
   4. Add 3 column volumes (CV) of PBS containing 20 mM of imidazole for washing, and collect the washing fraction.
   5. Gradient elute with 3 CV of PBS containing low to high concentrations (e.g., 0.3 M, 0.4 M, 0.5 M) of imidazole; elute 2x for each concentration.
   6. Use SDS-PAGE²¹ to identify the RBD-ST in different concentration gradients.

4. OMV-RBD bioconjugation and purification

1. Determine the protein concentration by the BCA method (see Table of Materials).
   1. Serially dilute the standard BSA protein solution from 2 mg/mL to 0.0625 mg/mL, dilute the purified OMV-SC and RBD-ST 10x, then mix BCA working solutions A and B (provided in the assay kit) at a ratio of 50:1 (v/v).
   2. Add diluted protein solution (25 µL/well) and mix with BCA working solution (200 µL/well); incubate at 37 °C for 30 min.
   3. Measure the absorbance (OD) at 562 nm of each well and calculate the protein concentration from the standard curve²².
2. Perform bioconjugation of OMV-SC and RBD-ST following the steps below.
   1. Mix OMV-SC and RBD-ST in PBS at a 40:1 (w/w) ratio.
   2. Vertically rotate to blend the mixture overnight at 15 rpm at 4 °C.
      NOTE: Different antigens may react in different proportions. One could try different reaction ratios based on the characteristics of the antigen.
3. Verify the reaction efficiency.
   1. Prepare 10 µL of OMV-SC, RBD-ST, and the reaction product (step 4.2.). Add 2.5 µL of 5x loading buffer (see Table of Materials), and heat the samples at 100 °C for 5 min. Load the samples into the gel (10 µL/well). Perform electrophoresis at 60 V for 20 min and change the condition to 120 V for 1 h.
   2. Transfer the protein from the gel to PVDF western blotting membranes (see Table of Materials) at 100 V for 70 min.
   3. Put the membrane into 5% non-fat powdered milk/TBST and shake for 1 h. Then, wash 3x with TBST for 5 min per wash with shaking.
   4. Put the membrane into 0.1% His-Tag antibody/TBST (see Table of Materials) and shake for 1 h, then wash 3x with TBST for 5 min per wash with shaking.
   5. Put the membrane into 0.02% anti-mouse IgG1 antibody (HRP)/TBST (see Table of Materials) and shake for 40 min, then wash 3x with TBST for 5 min per wash with shaking.
   6. Add enhanced chemiluminescence solution (see Table of Materials) and expose the membrane.
4. Perform purification of OMV-RBD.
   1. Dilute 1 mL of reacted OMV-RBD solution to 10 mL, and centrifuge the dilution at 150,000 x g at 4 °C for 2 h.
   2. Use a pipette to discard the supernatant and suspend the residue with 10 mL of PBS. Centrifuge the suspension at 150,000 x g at 4 °C for 2 h.
   3. Discard the supernatant and suspend the residue with 1 mL of PBS.
      ​NOTE: If the solubility of the antigen is much lower, the same separation effect can be achieved by size-exclusion chromatography¹⁹.

5. Characterization

1. Perform dynamic light scattering (DLS).
   1. Dilute the samples to a 100 µg/mL concentration and add 1 mL of sample into the sample cell.
   2. Choose "Size" for "Measurement type", "Protein" for "Sample material", "Water" for "Dispersant", "25 °C" for "Temperature", and then load samples and measure automatically²³.
2. Capture images using transmission electron microscopy (TEM).
   1. Dilute the samples to 100 µg/mL. Take a 200-mesh copper grid, add 20 µL of sample to it, and allow it to get absorbed for 10 min.
   2. Use filter paper to wick off the solution, add 20 µL of 3% uranyl acetate to the support film, and stain for 30 s.
   3. Wick off the uranyl acetate and dry the film naturally at room temperature for 1 h.
   4. Load the samples to the TEM system (see Table of Materials) and capture images at 80 kV.

Wyniki

The flowchart for this protocol is shown in Figure 1. This protocol could be a general approach to utilizing OMVs as a vaccine platform; one only needs to choose the appropriate expression systems based on the type of antigens.

Figure 2 provides a feasible plasmid design scheme. The SC gene is connected with the ClyA gene via a flexible linker, while ST connects to the 5' terminal of the RBD gene with a His-tag gene for purif...

Dyskusje

To create a "Plug-and-Display" nanoparticle vaccine platform, SC-fused ClyA was expressed in BL21(DE3) strains, which is one of the most widely used models for recombinant protein production because of its advantages in protein expression²⁴, so that there would be enough SC fragment displaying on the surface of the OMVs during the process of bacteria proliferation. At the same time, an ST-fused target antigen was prepared for the chemical coupling between the antigens and OMVs. This experi...

Materiały

Name	Company	Catalog Number	Comments
Ampicillin sodium	Sangon Biotech	A610028
Automated cell counter	Countstar	BioTech
BCA protein quantification Kit	cwbio	cw0014s
ChemiDoc Touching Imaging System	Bio-rad
Danamic Light Scattering	Malvern	Zetasizer Nano S90
Electrophoresis apparatus	Cavoy	Power BV
EZ-Buffers H 10X TBST Buffer	Sangon Biotech	C520009
Goat pAb to mouse IgG1	Abcam	ab97240
High speed freezing centrifuge	Bioridge	H2500R
His-Tag mouse mAb	Cell signaling technology	2366s
Imidazole	Sangon Biotech	A600277
Isopropyl beta-D-thiogalactopyranoside	Sangon Biotech	A600118
Ni-NTA His-Bind Superflow	Qiagen	70691
Non-fat powdered milk	Sangon Biotech	A600669
OPM-293 cell culture medium	Opm biosciences	81075-001
pcDNA3.1 RBD-ST plasmid	Wuhan genecreat biological techenology
Phosphate buffer saline	ZSGB-bio	ZLI-9061
Polyethylenimine Linear	Polysciences	23966-1
Prestained protein ladder	Thermo	26616
pThioHisA ClyA-SC plasmid	Wuhan genecreat biological techenology
PVDF Western Blotting Membranes	Roche	03010040001
Quixstand benchtop systems (100 kD hollow fiber column)	GE healthcare
SDS-PAGE loading buffer (5x)	Beyotime	P0015
Sodium chloride	Sangon Biotech	A100241
Supersignal west pico PLUS (enhanced chemiluminescence solution)	Thermo	34577
Suspension instrument	Life Technology	Hula Mixer
Transmission Electron Microscope	Hitachi	HT7800
Tryptone	Oxoid	LP0042B
Ultracentrifuge	Beckman coulter	XPN-100
Ultraviolet spectrophotometer	Hitachi	U-3900
Yeast extract	Sangon Biotech	A610961