Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

Abstract

Source: Feng, R., et al. A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain. J. Vis. Exp. (2022).

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), creating OMVs that display the antigen, serving as a multivalent vaccine.

Protocol

1. Plasmid construction Insert DNA encoding SpyCatcher sequence (Figure 1) into an ampicillin-resistant pThioHisA-Cytolysin A (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 report. Ligate the synthesized SpyTag-SARS-CoV-2 receptor-binding domain (RBD)-polyhistidine tag (Histag) fusion gene (Figure 1) into a pcDNA3.1 plasm…

Representative Results

Figure 1: Plasmid sequences used in the present study.

Materials

Ampicillin sodium	Sangon Biotech	A610028
Automated cell counter	Countstar	BioTech
BCA protein quantification Kit	cwbio	cw0014s
Electrophoresis apparatus	Cavoy	Power BV
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
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
Quixstand benchtop systems (100 kD hollow fiber column)	GE healthcare
SDS-PAGE loading buffer (5x)	Beyotime	P0015
Sodium chloride	Sangon Biotech	A100241
Suspension instrument	Life Technology	Hula Mixer
Tryptone	Oxoid	LP0042B
Ultracentrifuge	Beckman coulter	XPN-100
Ultraviolet spectrophotometer	Hitachi	U-3900
Yeast extract	Sangon Biotech	A610961