Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Summary

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Abstract

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Guérin (BCG) is safe and effective for protection against children's severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Introduction

Various strategies have been proposed to replace the current TB vaccine BCG, including protein adjuvant systems, viral vectored technologies, attenuated live M.tb strains, and genetically modified BCG strains, either to introduce genes over-expressing BCG antigens that are not sufficiently expressed during infection¹ or Mtb-specific antigens not present in BCG². Genetic engineering, however, faces many barriers including the uncertain level of safety, the time-consuming process, and the low efficiency of expression vectors^4,5. With regards to im....

Protocol

All animals were maintained in accordance with protocols approved by the Animal Care and Use Committees at the University of British Columbia. Experiments were approved by the Animal Care and Use Committees and performed according to the Canadian Council on Animal Care Guidelines. The animal assurance welfare number is A11-0247.

1. Generation of Monomeric Avidin Fusion Proteins Expressing Plasmids

1. Sub-clone monomeric avidin sequence¹² into pDEST17 plasmid between the "CTC" and "GAA" sites which corresponds to 133-134bp. (i.e., between the 6-histag and the pDEST17 Attr1 re....

Results

With the general procedures described above, the feasibility of BCG surface biotinylation and decoration with surrogate antigen OVA was examined. The immunogenicity of the modified BCG was then tested in vivo. The bacterial surface was easily labeled with biotin for rapid display of avidin chimeric antigens without any detectable changes in bacterial phenotypes. The resulting modified BCG is efficiently ingested by antigen presentation cells and can induce an OVA-specific immune .......

Discussion

We reported in this study a non-genetic approach for rapid and effective display of exogenous proteins on BCG surface to add either specific antigens or specific functional properties expected to efficiently improve the bacterium's immunogenicity. We demonstrated that the BCG cell surface could be easily biotinylated for instantaneous surface decoration with avidin fusion proteins. The total procedure can be performed within 2 h, while genetic transformation and selection of positive clones requires 2 to 3 months of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Endotoxin-free RPMI 1640	StemCell Technologies	36750
Sulfo-NHS SS biotin	Thermo Fisher	21328
FITC-conjugated streptavidin	Sigma-Aldrich	S3762
Phycoerythrin (PE)-conjugated I-Ab-OVA323-339 tetramer	MBL International	TS-M710-1
7-AAD	BD Pharmingen	559925
TALON polyhistidine-Tag purification resin	Clontech	635501
Alexa Fluor (AF) 647 conjugated rat anti-mouse CD4	BD Bioscience	557681
AF647 rat anti-mouse IFN-g	BD Bioscience	557735
AF647 rat anti-mouse I-A/I-E	BD Bioscience	562367
PeCy7 rat anti-mouse CD4	BD Bioscience	552775
PE rat anti-mouse CD8 Ab	BD Bioscience	561095
AF 647 rat anti-mouse H-2kb	BD Bioscience	562832
FITC-conjugated goat anti rabbit antibody	Thermo Fisher	31635
AF 647 rat anti-mouse CD4	BD Bioscience	557681
Ultra-small gold-conjugated goat anti-rabbit IgG	Electron Microscopy Sciences	25100
Middlebrook 7H9 broth	BD Diagnostic Systems	271310
OADC	BD Diagnostic Systems	B11886
Tween 80	Sigma-Aldrich	P1379
RAW 264.7 murine macrophage cell lines	American Type Culture Collection
pDEST17 plasmid	Invitrogen	11803012
pUC57-OVA plasmid	GenScript	SD1176
BP clonase	Invitrogen	11789020
LR clonase	Invitrogen	11791043
pDONR221 plasmid	Invitrogen	12536017
Ni-NTA columns	Qiagen	31014
Pierce protein concentrators	Thermo Fisher	88527
Flurosave	Calbiochem-Novabiochem	345789
Axioplan II epifluorescence microscope	Carl Zeiss Inc
CCD digital camera	Retiga EX, QImaging
Tecnai G2 200kV electron microscope	FEI Company	G2 200Kv
70μm Falcon cell strainer	Thermo Fisher	87712
EasyStep mouse biotin positive selection kit	StemCell	18556
biotin-Ter119/Ertyroid cells antibody	BioLegend	116203
Brefeldin A	BD Pharmingen	555029
Cytofix/Cytoperm kit	BD Pharmingen	554714
Bright-Glo Luciferase assay system	Promega	e2620
Turner Biosystem luminometer	Promega	TD-20/20
Leica EM UC6 microtome	Leica Microsystems	UC6
Novalyphe NL 500 freeze dryer	Savant Instruments	NL 50
Wheaton boroscilicate glass vials	Wheaton	VWR 66011-675