Limits...
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.

Show MeSH

Related in: MedlinePlus

Biotinylation of BCG surface does not affect its growth or its survival in the macrophages.(A) Biot-BCG and control unlabeled wild-type BCG were grown in complete 7H9 media and replication was monitored over an 8-day period. The results are expressed as growth curves, i.,e., absorbance at 600nm as function of time. (B) Macrophages were infected with Biot-BCG-Luc or control unlabeled BCG-Luc for the indicated time periods, and then cell lysates were prepared and assayed for bioluminescence to detect viable bacteria. Results are expressed as relative light units (RLU). Data shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696857&req=5

pone.0145833.g003: Biotinylation of BCG surface does not affect its growth or its survival in the macrophages.(A) Biot-BCG and control unlabeled wild-type BCG were grown in complete 7H9 media and replication was monitored over an 8-day period. The results are expressed as growth curves, i.,e., absorbance at 600nm as function of time. (B) Macrophages were infected with Biot-BCG-Luc or control unlabeled BCG-Luc for the indicated time periods, and then cell lysates were prepared and assayed for bioluminescence to detect viable bacteria. Results are expressed as relative light units (RLU). Data shown are representative of three independent experiments.

Mentions: We next examined the effect of bacterial surface modification, as result of biotinylation, on the growth kinetics in culture media. The result obtained showed that Biot-BCG displays a growth profile similar to that of control untreated bacteria, over a 8-day period (Fig 3A). On the other hand, we took advantage of our BCG strain expressing luciferase (BCG-Luc) [20] to examine Biot-BCG survival in the macrophage. Luminescence signals recorded (Fig 3B) showed that biotinylated and control bacteria display similar profiles of gradual viability decrease within the macrophage. Taking together, these data clearly demonstrate that surface modification with biotin is compatible with bacterial growth and more importantly, does not increase BCG persistence in the macrophage, which would be interpreted as a conversion into a virulent bacterium.


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)

Biotinylation of BCG surface does not affect its growth or its survival in the macrophages.(A) Biot-BCG and control unlabeled wild-type BCG were grown in complete 7H9 media and replication was monitored over an 8-day period. The results are expressed as growth curves, i.,e., absorbance at 600nm as function of time. (B) Macrophages were infected with Biot-BCG-Luc or control unlabeled BCG-Luc for the indicated time periods, and then cell lysates were prepared and assayed for bioluminescence to detect viable bacteria. Results are expressed as relative light units (RLU). Data shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145833.g003: Biotinylation of BCG surface does not affect its growth or its survival in the macrophages.(A) Biot-BCG and control unlabeled wild-type BCG were grown in complete 7H9 media and replication was monitored over an 8-day period. The results are expressed as growth curves, i.,e., absorbance at 600nm as function of time. (B) Macrophages were infected with Biot-BCG-Luc or control unlabeled BCG-Luc for the indicated time periods, and then cell lysates were prepared and assayed for bioluminescence to detect viable bacteria. Results are expressed as relative light units (RLU). Data shown are representative of three independent experiments.
Mentions: We next examined the effect of bacterial surface modification, as result of biotinylation, on the growth kinetics in culture media. The result obtained showed that Biot-BCG displays a growth profile similar to that of control untreated bacteria, over a 8-day period (Fig 3A). On the other hand, we took advantage of our BCG strain expressing luciferase (BCG-Luc) [20] to examine Biot-BCG survival in the macrophage. Luminescence signals recorded (Fig 3B) showed that biotinylated and control bacteria display similar profiles of gradual viability decrease within the macrophage. Taking together, these data clearly demonstrate that surface modification with biotin is compatible with bacterial growth and more importantly, does not increase BCG persistence in the macrophage, which would be interpreted as a conversion into a virulent bacterium.

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