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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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mAvidin-OVA binding to Biot-BCG, its stability and delivery inside macrophages.(A) Biot-dsRed-BCG and control non-biotinylated dsRed-BCG were mixed with Avi-OVA at room temperature for 1 h and the extent of OVA binding was evaluated by surface staining with rabbit anti-avidin Ab and FITC- goat anti-rabbit IgG (FL1). Sample were washed and analyzed by FACS. Results are presented as histogram of green fluorescence intensity with mean± SEM of mean fluorescence intensity (MFI) of Biot-BCG-AviOVA from three independent experiments. (B) Lyophilized Biot-BCG coated with Avi-OVA and freshly made Biot-BCG with Avi-OVA were labeled and analyzed by FACS as described in A. Data shown are representative of three independent experiments. Mean of MFI±SEM for biotinylated BCG coated with Avi-OVA are shown. (C) RAW macrophages were infected with OVA-dsRed-BCG or dsRed-BCG for 24 h at 37°C. Samples were washed, treated with trypsin and fixed and analyzed by FACS. Results are expressed as red fluorescence histogram, which reflect the extent of phagocytosis. Values indicated average percentage ±SEM of cells ingesting BCG. Results shown are representative of three independent experiments.
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pone.0145833.g004: mAvidin-OVA binding to Biot-BCG, its stability and delivery inside macrophages.(A) Biot-dsRed-BCG and control non-biotinylated dsRed-BCG were mixed with Avi-OVA at room temperature for 1 h and the extent of OVA binding was evaluated by surface staining with rabbit anti-avidin Ab and FITC- goat anti-rabbit IgG (FL1). Sample were washed and analyzed by FACS. Results are presented as histogram of green fluorescence intensity with mean± SEM of mean fluorescence intensity (MFI) of Biot-BCG-AviOVA from three independent experiments. (B) Lyophilized Biot-BCG coated with Avi-OVA and freshly made Biot-BCG with Avi-OVA were labeled and analyzed by FACS as described in A. Data shown are representative of three independent experiments. Mean of MFI±SEM for biotinylated BCG coated with Avi-OVA are shown. (C) RAW macrophages were infected with OVA-dsRed-BCG or dsRed-BCG for 24 h at 37°C. Samples were washed, treated with trypsin and fixed and analyzed by FACS. Results are expressed as red fluorescence histogram, which reflect the extent of phagocytosis. Values indicated average percentage ±SEM of cells ingesting BCG. Results shown are representative of three independent experiments.

Mentions: Initial experiments examined the extent and success of Biot-BCG surface decoration with exogenous proteins. Biot-dsRed-BCG and control non-biotinylated bacteria were exposed to OVA antigen peptide (Avi-OVA, 10 μg/ml) for 1 h at room temperature. Bacteria were then washed and bound OVA was detected with rabbit anti-avidin Ab. FACS analyses show a minimal binding of OVA peptide to non-biotinylated bacteria (MFI = 8.31 ± 0.42, Fig 4A top panel). In contrast, significant levels of OVA peptide were detected on the surface of Biot-BCG as reflected by total shift of fluorescence histograms corresponding to Avi-OVA coated Biot-BCG (MFI = 54.67±4.98) relative to control uncoated bacteria (MFI = 5.92±0.22, Fig 4A lower panel). These data demonstrated the efficiency and specificity of mAvidin fusion protein binding to the surface of biotinylated bacteria.


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)

mAvidin-OVA binding to Biot-BCG, its stability and delivery inside macrophages.(A) Biot-dsRed-BCG and control non-biotinylated dsRed-BCG were mixed with Avi-OVA at room temperature for 1 h and the extent of OVA binding was evaluated by surface staining with rabbit anti-avidin Ab and FITC- goat anti-rabbit IgG (FL1). Sample were washed and analyzed by FACS. Results are presented as histogram of green fluorescence intensity with mean± SEM of mean fluorescence intensity (MFI) of Biot-BCG-AviOVA from three independent experiments. (B) Lyophilized Biot-BCG coated with Avi-OVA and freshly made Biot-BCG with Avi-OVA were labeled and analyzed by FACS as described in A. Data shown are representative of three independent experiments. Mean of MFI±SEM for biotinylated BCG coated with Avi-OVA are shown. (C) RAW macrophages were infected with OVA-dsRed-BCG or dsRed-BCG for 24 h at 37°C. Samples were washed, treated with trypsin and fixed and analyzed by FACS. Results are expressed as red fluorescence histogram, which reflect the extent of phagocytosis. Values indicated average percentage ±SEM of cells ingesting BCG. Results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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pone.0145833.g004: mAvidin-OVA binding to Biot-BCG, its stability and delivery inside macrophages.(A) Biot-dsRed-BCG and control non-biotinylated dsRed-BCG were mixed with Avi-OVA at room temperature for 1 h and the extent of OVA binding was evaluated by surface staining with rabbit anti-avidin Ab and FITC- goat anti-rabbit IgG (FL1). Sample were washed and analyzed by FACS. Results are presented as histogram of green fluorescence intensity with mean± SEM of mean fluorescence intensity (MFI) of Biot-BCG-AviOVA from three independent experiments. (B) Lyophilized Biot-BCG coated with Avi-OVA and freshly made Biot-BCG with Avi-OVA were labeled and analyzed by FACS as described in A. Data shown are representative of three independent experiments. Mean of MFI±SEM for biotinylated BCG coated with Avi-OVA are shown. (C) RAW macrophages were infected with OVA-dsRed-BCG or dsRed-BCG for 24 h at 37°C. Samples were washed, treated with trypsin and fixed and analyzed by FACS. Results are expressed as red fluorescence histogram, which reflect the extent of phagocytosis. Values indicated average percentage ±SEM of cells ingesting BCG. Results shown are representative of three independent experiments.
Mentions: Initial experiments examined the extent and success of Biot-BCG surface decoration with exogenous proteins. Biot-dsRed-BCG and control non-biotinylated bacteria were exposed to OVA antigen peptide (Avi-OVA, 10 μg/ml) for 1 h at room temperature. Bacteria were then washed and bound OVA was detected with rabbit anti-avidin Ab. FACS analyses show a minimal binding of OVA peptide to non-biotinylated bacteria (MFI = 8.31 ± 0.42, Fig 4A top panel). In contrast, significant levels of OVA peptide were detected on the surface of Biot-BCG as reflected by total shift of fluorescence histograms corresponding to Avi-OVA coated Biot-BCG (MFI = 54.67±4.98) relative to control uncoated bacteria (MFI = 5.92±0.22, Fig 4A lower panel). These data demonstrated the efficiency and specificity of mAvidin fusion protein binding to the surface of biotinylated bacteria.

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