Name: A Plug-And-Display Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain (Video) | JoVE Journal
Uploaded: 2022-07-25T14:30:00.000+00:00
Duration: 8 min 7 s
Description: Learn to bioengineer outer membrane vesicles as a versatile "Plug-and-Display" vaccine platform for enhanced antigen presentation. This method enables effective immune responses through surface antigen display.

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5 minutes

This work was supported by the Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and the Chinese National Natural Science Foundation Project (No. 31670936, 82041045).

1. Plasmid construction
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
2. OMV-SC preparation
<ol>
	<li>Perform ClyA-SC transformation following the steps below.
	<ol>
		<li>Add 5 &#181;L of pThioHisA ClyA-SC plasmid solution (50 ng/&#181;L) to 50 &#181;L of BL21 competent strain, gently blow, and let cool on ice for 30 min.</li>
		<li>Place the solution for 90 s in the water bath at 42 &#176;C, and then immediately put the mixed solution on ice for 3 min.</li>
		<li>Add 500 &#181;L of LB medium to the bacterial suspension and, after mixing, culture at 220 rpm at 37 &#176;C for 1 h.</li>
		<li>Plate all the transformation onto an LB agar plate containing ampicillin (100 &#181;g/mL, see Table of Materials) and culture overnight at 37 &#176;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.</li>
	</ol>
	</li>
	<li>For OMV-SC production, perform the steps below.
	<ol>
		<li>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 &#176;C.</li>
		<li>Inoculate the bacterial solution (from step 2.2.1.) into 2 L medium, culture at 220 rpm, 37 &#176;C for 5 h until the logarithmic growth stage (the OD600nm is between 0.6 to 0.8).</li>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>Perform OMV-SC purification.
	<ol>
		<li>Centrifuge the bacterial solution at 7,000 x g at 4 &#176;C for 30 min.</li>
		<li>Filter the supernatant with a 0.22 &#181;m membrane filter, then concentrate it using a 100 kD ultrafiltration membrane or hollow fiber column (see Table of Materials).</li>
		<li>Filter the concentrate through a 0.22 &#181;m membrane filter, then centrifuge it at 150,000 x g at 4 &#176;C for 2 h using an ultracentrifuge (see Table of Materials), and discard the supernatant with a pipette.</li>
		<li>Resuspend the precipitation with PBS and store it at &#8722;80 &#176;C. The solution can maintain long-term stability at &#8722;80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. RBD-ST preparation
<ol>
	<li>Perform RBD-ST transfection following the steps below.
	<ol>
		<li>Select an appropriate eukaryotic expression system (e.g., HEK293F) and culture the cells overnight at 130 rpm at 37 &#176;C after recovery.</li>
		<li>Add 20 &#181;L 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 &#176;C for 4 h.</li>
		<li>Filter RBD-ST plasmid through a 0.22 &#181;m membrane filter, and add 300 &#181;g of plasmids into the cell culture medium (see Table of Materials) until the final volume is 10 mL; shake for 10 s.</li>
		<li>Heat PEI (1 mg/mL, see Table of Materials) to 65 &#176;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.</li>
		<li>Add plasmid solution to the PEI solution, shake the mixture intermittently for 10 s, and incubate it at 37 &#176;C for 15 min.</li>
		<li>Add the mixture to 280 mL of cell culture medium and culture at 130 rpm at 37 &#176;C for 5 days.</li>
	</ol>
	</li>
	<li>Perform RBD-ST purification.
	<ol>
		<li>Centrifuge the cells at 6,000 x g at 25 &#176;C for 20 min and use a pipette to collect the supernatant.</li>
		<li>Fill the column with 2 mL of Ni-NTA agarose (see Table of Materials) and wash it 3x with 3x PBS.</li>
		<li>Add imidazole (see Table of Materials) into the cell supernatant to make the final concentration of 20 mM, and load the cell supernatant 2x.</li>
		<li>Add 3 column volumes (CV) of PBS containing 20 mM of imidazole for washing, and collect the washing fraction.</li>
		<li>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.</li>
		<li>Use SDS-PAGE21 to identify the RBD-ST in different concentration gradients.</li>
	</ol>
	</li>
</ol>
4. OMV-RBD bioconjugation and purification
<ol>
	<li>Determine the protein concentration by the BCA method (see Table of Materials).
	<ol>
		<li>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).</li>
		<li>Add diluted protein solution (25 &#181;L/well) and mix with BCA working solution (200 &#181;L/well); incubate at 37 &#176;C for 30 min.</li>
		<li>Measure the absorbance (OD) at 562 nm of each well and calculate the protein concentration from the standard curve22.</li>
	</ol>
	</li>
	<li>Perform bioconjugation of OMV-SC and RBD-ST following the steps below.
	<ol>
		<li>Mix OMV-SC and RBD-ST in PBS at a 40:1 (w/w) ratio.</li>
		<li>Vertically rotate to blend the mixture overnight at 15 rpm at 4 &#176;C. 
		NOTE: Different antigens may react in different proportions. One could try different reaction ratios based on the characteristics of the antigen.</li>
	</ol>
	</li>
	<li>Verify the reaction efficiency.
	<ol>
		<li>Prepare 10 &#181;L of OMV-SC, RBD-ST, and the reaction product (step 4.2.). Add 2.5 &#181;L of 5x loading buffer (see Table of Materials), and heat the samples at 100 &#176;C for 5 min. Load the samples into the gel (10 &#181;L/well). Perform electrophoresis at 60 V for 20 min and change the condition to 120 V for 1 h.</li>
		<li>Transfer the protein from the gel to PVDF western blotting membranes (see Table of Materials) at 100 V for 70 min.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Add enhanced chemiluminescence solution (see Table of Materials) and expose the membrane.</li>
	</ol>
	</li>
	<li>Perform purification of OMV-RBD.
	<ol>
		<li>Dilute 1 mL of reacted OMV-RBD solution to 10 mL, and centrifuge the dilution at 150,000 x g at 4 &#176;C for 2 h.</li>
		<li>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 &#176;C for 2 h.</li>
		<li>Discard the supernatant and suspend the residue with 1 mL of PBS. 
		&#8203;NOTE: If the solubility of the antigen is much lower, the same separation effect can be achieved by size-exclusion chromatography19.</li>
	</ol>
	</li>
</ol>
5. Characterization
<ol>
	<li>Perform dynamic light scattering (DLS).
	<ol>
		<li>Dilute the samples to a 100 &#181;g/mL concentration and add 1 mL of sample into the sample cell.</li>
		<li>Choose &#34;Size&#34; for &#34;Measurement type&#34;, &#34;Protein&#34; for &#34;Sample material&#34;, &#34;Water&#34; for &#34;Dispersant&#34;, &#34;25 &#176;C&#34; for &#34;Temperature&#34;, and then load samples and measure automatically23.</li>
	</ol>
	</li>
	<li>Capture images using transmission electron microscopy (TEM).
	<ol>
		<li>Dilute the samples to 100 &#181;g/mL. Take a 200-mesh copper grid, add 20 &#181;L of sample to it, and allow it to get absorbed for 10 min.</li>
		<li>Use filter paper to wick off the solution, add 20 &#181;L of 3% uranyl acetate to the support film, and stain for 30 s.</li>
		<li>Wick off the uranyl acetate and dry the film naturally at room temperature for 1 h.</li>
		<li>Load the samples to the TEM system (see Table of Materials) and capture images at 80 kV.</li>
	</ol>
	</li>
</ol>

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<li>Balhuizen, M. D., Veldhuizen, E. J. A., Haagsman, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Outer+membrane+vesicle+induction+and+isolation+for+vaccine+development%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Outer membrane vesicle induction and isolation for vaccine development.</a> Frontiers in Microbiology. 12, 629090(2021).</li>
<li>Hua, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+immunomodulator+delivery+platform+based+on+bacterial+biomimetic+vesicles+for+enhanced+antitumor+immunity%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">A novel immunomodulator delivery platform based on bacterial biomimetic vesicles for enhanced antitumor immunity.</a> Advanced Materials. 33 (43), 2103923(2021).</li>
</ol>

The authors have nothing to disclose.

To create a &#34;Plug-and-Display&#34; 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...

As a potential vaccine platform, outer membrane vesicles (OMVs) have attracted more and more attention in recent years1,2. 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 cell11,12,13. Thus, RBD seems to be a prime target for vaccine discovery14,15,16. 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.

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a "plug-and-display" nanoparticle vaccine platform based on outer membrane vesicles displaying sars-cov-2 receptor-binding domain

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

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&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; 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.

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 purification a...

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

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

a-plug-display-nanoparticle-vaccine-platform-based-on-outer-membrane

Research

JoVE Journal

Bioengineering

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay. <br...

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

This video demonstrates a method to generate multivalent-displaying outer membrane vesicle (OMV) vaccines. Mammalian cells are transfected to express and secrete viral antigens fused to a SpyTag and a polyhistidine tag. These antigens are then purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column. The purified antigen is mixed with outer membrane vesicles expressing surface-bound SpyCatcher (SC) protein. Multiple antigens covalently attach to SpyCatchers via their SpyTags (ST),...

Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Engineering Antiviral Agents via Surface Plasmon Resonance

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

We present an optimized in vitro protocol for producing SARS-CoV-2 virus-like particles that closely mimic the authentic virus. This approach enables the investigation of viral infection, assembly, and egress mechanisms without the constraints of requiring a biosafety level 3 laboratory.

Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro

This article describes the methodology for generating SARS-CoV-2 pseudotyped particles using a luciferase reporter system and its use to identify entry inhibitors. These protocols are demonstrated using two different entry-inhibitors, a recombinant human ACE2-Fc fusion protein and the small-molecule compound arbidol.

Procedures for the Identification of SARS-CoV-2 Entry Inhibitors as Potential Antivirals using MLV-Based Pseudoviruses

Learn to bioengineer outer membrane vesicles as a versatile "Plug-and-Display" vaccine platform for enhanced antigen presentation. This method enables effective immune responses through surface antigen display.

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

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