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

Summary

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

Abstract

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.

Introduction

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 en....

Protocol

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.......

Discussion

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.......

Materials

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