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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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 experimental scheme&#39;s advantages and future application prospects are mainly reflected in three aspects. First, the &#34;Plug-and-Display&#34; system of different antigens and biological nanoparticles is realized. The reaction and purification process could be fast and straightforward, which has significant advantages in developing vaccines for emergent infectious diseases and other scenarios. Second, it achieves the goal of high-density antigen displaying and provides a vaccine design method for some antigens with low immunogenicity. Third, the individual expression of OMVs and RBD (antigenic protein) is beneficial to ensure high antigen activity because conventional prokaryotic expression systems lack functions such as particular cofactors, molecular chaperones, and post-translational modifications, which may lead to protein loss and misfolding.
In the process of plasmid construction, the ClyA was chosen as a target to connect with SC mainly due to the loose barrel-shaped structure of the C-terminal of ClyA. Studies have shown that the hemoglobin protease (Hbp) autotransporter can display antigens from Streptococcus pneumonia and Mycobacterium tuberculosis25,26. Otherwise, if the antigen to be displayed is from a prokaryote, it is possible to construct the antigens directly into the C-terminal of ClyA without using SC/ST conjugation. This method may result in OMVs with higher displaying density, but there is still a risk that the antigenic proteins will not fold correctly. It is important to note that a flexible linker needs to be designed and used when SC/ST is inserted into the target fragment, which helps to improve the reaction efficiency between SC and ST. The linker sequence used here was GSGGSGGSGTG, and other flexible linkers can also be selected.
In addition to the SC/ST system for click-chemistry conjugation, Snooptag/Snoopcatcher and Sortase A conjugation are also commonly used for covalent conjugation between proteins27,28. At the same time, it has also been reported that the introduction of multiple different linkage systems on the same vector29 or the use of the same linkage system for multiple antigens19 can achieve the purpose of displaying multiple heterologous antigens on the same vector, and then polyvalent or multifunctional vaccines can be prepared.
High cytotoxicity and low yield limit the widespread use of OMVs as vaccine platforms4,30. Some LPS-related genes have been knocked out in the strain used in this protocol, according to relevant literature31,32,33, in order to reduce the cytotoxicity of OMVs. 1.18 mg of OMVs can be extracted from each 1 L of the bacterial solution by the method introduced in this protocol. The yield is still lower than that of some genetically modified high-vesicle-producing strains34. If necessary, high-pressure homogenization can also promote the germination of the bacterial membrane to produce more OMVs, which can achieve improved yield and displaying density at the same time35.
The induction conditions are crucial for the density of SC displayed on the surface of the OMVs. We monitored the expression of the target protein through SDS-PAGE and then screened out the induction condition, which is relatively mild and expresses more protein. Different proteins may require different induction conditions, and changes in the induction conditions may lead to differences in the density of SC displayed on the surface of the OMVs. Adjusting the antigen and carrier ratio after the experimental conditions change to explore the most appropriate reaction ratio is suggested. In addition, the purification of OMV-RBD can be achieved by SEM or ultracentrifugation. Both methods were tried; ultracentrifugation is more convenient, and the resulting OMV had a purity similar to the SEM protocol.

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.

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

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&#160;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&#39; terminal of the RBD gene with a His-tag gene for purification and verification. Western blot showed that the reaction gradually completes with increasing OMV-SC (Figure 3A). After ultracentrifugation, almost all of the unreacted RBD-ST remained in the supernatant. The second ultracentrifugation did not provide a further significant advantage over the first time (Figure 3B).
The distribution of particle size was determined by DLS (Figure 4). The Z-average hydrodynamic diameter of OMV-SC was 133 nm, while it was 152.6 nm for OMV-RBD (Table 1). These differences may be because RBD increases OMV particle size after intensive displaying. The results of TEM (Figure 5) are consistent with the DLS results. The images showed that the OMVs always have a standard spherical structure, whether connected to RBD or not. This indicates that the extraction, reaction, and purification conditions are conducive to maintaining the biological activity of OMVs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64213/64213fig01.jpg" /> 
Figure 1: Flowchart of OMV-RBD preparation. The ClyA-SC plasmid was transformed into E.coli, while the RBD-ST plasmid was transfected into HEK293F cells. After a period of culturing, OMV-SC and RBD-ST were isolated and purified. After bioconjugation in conventional buffer and ultracentrifugation, OMV-RBD was obtained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64213/64213fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64213/64213fig02.jpg" /> 
Figure 2: The plasmid profiles used in this study. (A) pThioHisA ClyA-SC plasmid. (B)pcDNA RBD-ST plasmid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64213/64213fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64213/64213fig03.jpg" /> 
Figure 3: Verification of OMV-RBD by western blot. (A) The exploration of the reaction ratio between OMV-SC and RBD-ST with anti-6x HisTag. M: molecular weight marker 1: OMV-SC; 2: RBD-ST; 3: OMV:RBD = 10:1 (w/w); 4: OMV:RBD = 20:1; 5: OMV:RBD = 40:1.&#160;(B)&#160;Validation of the efficiency of ultracentrifugation with anti-6x HisTag. M: molecular weight marker 1: OMV-SC; 2: OMV-RBD pre-ultracentrifugation; 3: RBD-ST; 4: supernatant post first ultracentrifugation; 5: precipitate post first ultracentrifugation; 6: supernatant post second ultracentrifugation; 7: precipitate post second ultracentrifugation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64213/64213fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64213/64213fig04.jpg" /> 
Figure 4: Particle size distribution measured by DLS. (A) OMV-SC. (B) OMV-RBD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64213/64213fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64213/64213fig05.jpg" /> 
Figure 5: Visualization of OMVs by TEM. (A) OMV-SC (200 &#181;g/mL). (B) OMV-RBD (200 &#181;g/mL). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64213/64213fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample Name</td>
			<td>Temperature (&#176;C)</td>
			<td>Z-Ave (d.nm)</td>
			<td>PdI</td>
		</tr>
		<tr>
			<td>OMV-SC</td>
			<td>25</td>
			<td>133</td>
			<td>0.483</td>
		</tr>
		<tr>
			<td>OMV-RBD</td>
			<td>24.9</td>
			<td>152.6</td>
			<td>0.569</td>
		</tr>
	</tbody>
</table>
Table 1: Parameters measured by DLS.
Supplementary File 1: Plasmid sequences used in the present study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64213/Supplementary document.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.4K Views.  Third Military Medical University. The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

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

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

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

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.

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Immunology and Infection

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

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.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

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

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

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

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

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

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

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

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.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

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.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...

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

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

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

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.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

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

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

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.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.

This article describes the protocols used to produce a novel vaccine delivery platform, &#34;polybubbles,&#34; to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...

Biology

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

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

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.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

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

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Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

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

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

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

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