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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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Avidin-fusion antigen co-localizes with MHC class II and class I molecules.Adherent BMA cells were infected with OVA decorated GFP-BCG for 4 h and then simulated with IFN-γ for 24 h. Cells were then fixed/permeabilized and stained with rabbit anti-avidin Ab, Alexa 594 anti-rabbit IgG and either Alexa 647 rat anti-mouse I-A (A) or Alexa 647 rat anti mouse H-2kb (B). Samples were mounted on microscope slides and analyzed by digital confocal microscopy. Green signal indicates the position of BCG-GFP and red signal reflects the localization of Avi-OVA. Blue signal indicates the position of MHC class II or class I molecules. Dotted line indicates area of interest. Arrowheads indicated Avi-OVA colocalization with MHC molecules. Images shown are representatives of two independent experiments.
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pone.0145833.g006: Avidin-fusion antigen co-localizes with MHC class II and class I molecules.Adherent BMA cells were infected with OVA decorated GFP-BCG for 4 h and then simulated with IFN-γ for 24 h. Cells were then fixed/permeabilized and stained with rabbit anti-avidin Ab, Alexa 594 anti-rabbit IgG and either Alexa 647 rat anti-mouse I-A (A) or Alexa 647 rat anti mouse H-2kb (B). Samples were mounted on microscope slides and analyzed by digital confocal microscopy. Green signal indicates the position of BCG-GFP and red signal reflects the localization of Avi-OVA. Blue signal indicates the position of MHC class II or class I molecules. Dotted line indicates area of interest. Arrowheads indicated Avi-OVA colocalization with MHC molecules. Images shown are representatives of two independent experiments.

Mentions: BMA3.1A7, a mouse macrophage cell line commonly used to study antigen presentation [22] was infected with GFP-BCG surface decorated with Avi-OVA and subjected to intracellular staining for OVA peptide and I-A. Confocal images obtained show that Avi-OVA co-localize with I-A (Fig 6A) molecules suggesting that avidin fusion antigen molecules dissociated from the surface of biotinylated bacteria and translocated to compartments specialized for antigen processing and loading into MHC class II molecules. On the other hand, intracellular staining for H-2kb showed abundant co-localization of class I molecules and OVA peptide within BCG phagosomes (Fig 6B), suggestive of potential presentation of OVA antigen to CD8+ T cells by the macrophage, consistent with previous studies showing that phagosomes are competent organelles for antigen cross-presentation [23].


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)

Avidin-fusion antigen co-localizes with MHC class II and class I molecules.Adherent BMA cells were infected with OVA decorated GFP-BCG for 4 h and then simulated with IFN-γ for 24 h. Cells were then fixed/permeabilized and stained with rabbit anti-avidin Ab, Alexa 594 anti-rabbit IgG and either Alexa 647 rat anti-mouse I-A (A) or Alexa 647 rat anti mouse H-2kb (B). Samples were mounted on microscope slides and analyzed by digital confocal microscopy. Green signal indicates the position of BCG-GFP and red signal reflects the localization of Avi-OVA. Blue signal indicates the position of MHC class II or class I molecules. Dotted line indicates area of interest. Arrowheads indicated Avi-OVA colocalization with MHC molecules. Images shown are representatives of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145833.g006: Avidin-fusion antigen co-localizes with MHC class II and class I molecules.Adherent BMA cells were infected with OVA decorated GFP-BCG for 4 h and then simulated with IFN-γ for 24 h. Cells were then fixed/permeabilized and stained with rabbit anti-avidin Ab, Alexa 594 anti-rabbit IgG and either Alexa 647 rat anti-mouse I-A (A) or Alexa 647 rat anti mouse H-2kb (B). Samples were mounted on microscope slides and analyzed by digital confocal microscopy. Green signal indicates the position of BCG-GFP and red signal reflects the localization of Avi-OVA. Blue signal indicates the position of MHC class II or class I molecules. Dotted line indicates area of interest. Arrowheads indicated Avi-OVA colocalization with MHC molecules. Images shown are representatives of two independent experiments.
Mentions: BMA3.1A7, a mouse macrophage cell line commonly used to study antigen presentation [22] was infected with GFP-BCG surface decorated with Avi-OVA and subjected to intracellular staining for OVA peptide and I-A. Confocal images obtained show that Avi-OVA co-localize with I-A (Fig 6A) molecules suggesting that avidin fusion antigen molecules dissociated from the surface of biotinylated bacteria and translocated to compartments specialized for antigen processing and loading into MHC class II molecules. On the other hand, intracellular staining for H-2kb showed abundant co-localization of class I molecules and OVA peptide within BCG phagosomes (Fig 6B), suggestive of potential presentation of OVA antigen to CD8+ T cells by the macrophage, consistent with previous studies showing that phagosomes are competent organelles for antigen cross-presentation [23].

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