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Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

Liao TY, Lau A, Joseph S, Hytönen V, Hmama Z - PLoS ONE (2015)

Bottom Line: Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell.We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses.This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine and Vancouver Costal Health Research Institute, University of British Columbia, Vancouver, BC, Canada.

ABSTRACT
Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

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Efficiency of BCG biotinylation.(A) SSC and FL2 parameters and red fluorescent BCG were used to allow for proper gating of true bacteria during FACS analyses. (B) dsRed-BCG was biotinylated with various concentration of NHS-SS-biotin for 30 min at room temperature, then labeled with FITC-streptavidin and analyzed by FACS. Results are presented as histograms of green fluorescence intensity and the insert graph represents the mean±SEM of mean fluorescence intensity (MFI) deducted from 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.
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pone.0145833.g002: Efficiency of BCG biotinylation.(A) SSC and FL2 parameters and red fluorescent BCG were used to allow for proper gating of true bacteria during FACS analyses. (B) dsRed-BCG was biotinylated with various concentration of NHS-SS-biotin for 30 min at room temperature, then labeled with FITC-streptavidin and analyzed by FACS. Results are presented as histograms of green fluorescence intensity and the insert graph represents the mean±SEM of mean fluorescence intensity (MFI) deducted from 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Mentions: To examine the efficiency of BCG surface biotinylation we labeled bacteria expressing red fluorescence (dsRed-BCG) with various concentrations of Sulfo-NHS-SS-Biotin (0-1mM) and measured the relative abundance of bound biotin by staining with FITC-conjugated streptavidin and FACS analysis. The use of fluorescent bacteria allows for discriminating true bacteria from small-size contaminating particles in most FACS running buffers, which coincide with dots corresponding to BCG particles in SSC vs FSC displays (Fig 2A). Fluorescent histograms deducted from FACS analyses showed a direct relationship between Sulfo-NHS-SS-Biotin concentration and the level of biotin detected on the bacterial surface (Fig 2B). A total shift of fluorescence histogram (i.e. ~100% positive events) was observed in bacterial samples labeled with 0.5mM biotin and this concentration was used to generate biotinylated (Biot)-BCG throughout this study.


Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

Liao TY, Lau A, Joseph S, Hytönen V, Hmama Z - PLoS ONE (2015)

Efficiency of BCG biotinylation.(A) SSC and FL2 parameters and red fluorescent BCG were used to allow for proper gating of true bacteria during FACS analyses. (B) dsRed-BCG was biotinylated with various concentration of NHS-SS-biotin for 30 min at room temperature, then labeled with FITC-streptavidin and analyzed by FACS. Results are presented as histograms of green fluorescence intensity and the insert graph represents the mean±SEM of mean fluorescence intensity (MFI) deducted from 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696857&req=5

pone.0145833.g002: Efficiency of BCG biotinylation.(A) SSC and FL2 parameters and red fluorescent BCG were used to allow for proper gating of true bacteria during FACS analyses. (B) dsRed-BCG was biotinylated with various concentration of NHS-SS-biotin for 30 min at room temperature, then labeled with FITC-streptavidin and analyzed by FACS. Results are presented as histograms of green fluorescence intensity and the insert graph represents the mean±SEM of mean fluorescence intensity (MFI) deducted from 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.
Mentions: To examine the efficiency of BCG surface biotinylation we labeled bacteria expressing red fluorescence (dsRed-BCG) with various concentrations of Sulfo-NHS-SS-Biotin (0-1mM) and measured the relative abundance of bound biotin by staining with FITC-conjugated streptavidin and FACS analysis. The use of fluorescent bacteria allows for discriminating true bacteria from small-size contaminating particles in most FACS running buffers, which coincide with dots corresponding to BCG particles in SSC vs FSC displays (Fig 2A). Fluorescent histograms deducted from FACS analyses showed a direct relationship between Sulfo-NHS-SS-Biotin concentration and the level of biotin detected on the bacterial surface (Fig 2B). A total shift of fluorescence histogram (i.e. ~100% positive events) was observed in bacterial samples labeled with 0.5mM biotin and this concentration was used to generate biotinylated (Biot)-BCG throughout this study.

Bottom Line: Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell.We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses.This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine and Vancouver Costal Health Research Institute, University of British Columbia, Vancouver, BC, Canada.

ABSTRACT
Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Show MeSH
Related in: MedlinePlus