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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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In vivo CD4+ T cell response to ESAT6-decorated BCG and frequencies of T cells releasing cytokines in response to ESAT6.C57BL/6 mice were immunized with PBS, BCG WT alone or BCG WT surface decorated with Avi-ESAT6 as described in Fig 7. (A) Splenocytes from immunized animals were stained with PE-conjugated I-Ab-ESAT61-20 tetramers followed by AF647-CD4 Ab and 7-AAD. Samples were then analyzed by FACS. Results are expressed as two-parameter dot plots that show the average frequencies± SEM of tetramer positive events in the CD4+ population from two animals/group. Data shown are representative of three independent experiments. The data in graphs (B) are expressed as mean values of the absolute number± SEM of IFN-γ releasing CD4+ T cells (left graph) or IFN-γ releasing CD8+T cells (right graph) from two animals/group and three independent experiments. (C) Splenocytes from immunized animals were stimulated with recombinant ESAT6 protein then stained and analyzed by FACS as described in Fig 8. Results are expressed as two-parameter dot plots to show the average frequencies± SEM of IFN-γ producing cell subsets in CD4+ (upper panel) and CD8+ (lower panel) populations from two animals/group. Data shown are representative of three independent experiments. (D) Each column in graph represents the mean of absolute number± SEM of IFN-γ releasing CD4+ (Left graph) or CD8+T cells (right graph) from two animals/group and three independent experiments. *P<0.05; **P<0.01; ***P<0.001.
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pone.0145833.g009: In vivo CD4+ T cell response to ESAT6-decorated BCG and frequencies of T cells releasing cytokines in response to ESAT6.C57BL/6 mice were immunized with PBS, BCG WT alone or BCG WT surface decorated with Avi-ESAT6 as described in Fig 7. (A) Splenocytes from immunized animals were stained with PE-conjugated I-Ab-ESAT61-20 tetramers followed by AF647-CD4 Ab and 7-AAD. Samples were then analyzed by FACS. Results are expressed as two-parameter dot plots that show the average frequencies± SEM of tetramer positive events in the CD4+ population from two animals/group. Data shown are representative of three independent experiments. The data in graphs (B) are expressed as mean values of the absolute number± SEM of IFN-γ releasing CD4+ T cells (left graph) or IFN-γ releasing CD8+T cells (right graph) from two animals/group and three independent experiments. (C) Splenocytes from immunized animals were stimulated with recombinant ESAT6 protein then stained and analyzed by FACS as described in Fig 8. Results are expressed as two-parameter dot plots to show the average frequencies± SEM of IFN-γ producing cell subsets in CD4+ (upper panel) and CD8+ (lower panel) populations from two animals/group. Data shown are representative of three independent experiments. (D) Each column in graph represents the mean of absolute number± SEM of IFN-γ releasing CD4+ (Left graph) or CD8+T cells (right graph) from two animals/group and three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Mentions: Experiments using the surrogate antigen OVA clearly demonstrated the efficiency of surface decorated BCG to induce specific immune response in vivo. To validate this approach for Mtb specific proteins, we examined mouse immune response to BCG surface decorated with the early secreted Mtb antigen ESAT6, which is a major immunodominant antigen not expressed in BCG [11] and have been previously shown to induce protective T cell response against virulent Mtb [26]. Data from tetramer staining with PE-conjugated I-Ab-ESAT61-20 tetramer (Fig 9A) show that splenocytes from BCG-Avi-ESAT6 immunized mice contained a substantial ESAT6 specific CD4+ T cell subset (0.187±0.032% tetramer positive events) relative to the background value (0.052±0.004%) obtained from splenocytes from wild-type BCG immunized mice. This is also shown in the absolute number of ESAT6 specific CD4+ T cell numbers (Fig 9B). On the other hand, ICS showed significant expansion of IFN-γ producing CD4+ T cells in response to ESAT6 antigen in splenocytes isolated from BCG-Avi-ESAT6 (0.156±0.067%) compared to BCG WT (0.028±0.006%) vaccinated animals (Fig 9C top panel and Fig 9D top graph). Furthermore, the frequencies of IFN-γ producing CD8+ T cells in BCG-Avi-ESAT6 immunized mice were significantly greater than in mice immunized with wild-type BCG (Fig 9C bottom panel and Fig 9D bottom graph). Mice immunized with BCG coated with Avi-ESAT6 were able to induce 0.705±0.065% ESAT6 specific IFN-γ producing CD8+ T cells compared to 0.024±0.006% in BCG WT immunized mice. Taken together, these data showed that BCG surface coated with Mtb specific protein, ESAT6, was able to successfully induce specific ESAT6 immune response in vivo and improve immunogenicity of BCG.


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)

In vivo CD4+ T cell response to ESAT6-decorated BCG and frequencies of T cells releasing cytokines in response to ESAT6.C57BL/6 mice were immunized with PBS, BCG WT alone or BCG WT surface decorated with Avi-ESAT6 as described in Fig 7. (A) Splenocytes from immunized animals were stained with PE-conjugated I-Ab-ESAT61-20 tetramers followed by AF647-CD4 Ab and 7-AAD. Samples were then analyzed by FACS. Results are expressed as two-parameter dot plots that show the average frequencies± SEM of tetramer positive events in the CD4+ population from two animals/group. Data shown are representative of three independent experiments. The data in graphs (B) are expressed as mean values of the absolute number± SEM of IFN-γ releasing CD4+ T cells (left graph) or IFN-γ releasing CD8+T cells (right graph) from two animals/group and three independent experiments. (C) Splenocytes from immunized animals were stimulated with recombinant ESAT6 protein then stained and analyzed by FACS as described in Fig 8. Results are expressed as two-parameter dot plots to show the average frequencies± SEM of IFN-γ producing cell subsets in CD4+ (upper panel) and CD8+ (lower panel) populations from two animals/group. Data shown are representative of three independent experiments. (D) Each column in graph represents the mean of absolute number± SEM of IFN-γ releasing CD4+ (Left graph) or CD8+T cells (right graph) from two animals/group and three independent experiments. *P<0.05; **P<0.01; ***P<0.001.
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pone.0145833.g009: In vivo CD4+ T cell response to ESAT6-decorated BCG and frequencies of T cells releasing cytokines in response to ESAT6.C57BL/6 mice were immunized with PBS, BCG WT alone or BCG WT surface decorated with Avi-ESAT6 as described in Fig 7. (A) Splenocytes from immunized animals were stained with PE-conjugated I-Ab-ESAT61-20 tetramers followed by AF647-CD4 Ab and 7-AAD. Samples were then analyzed by FACS. Results are expressed as two-parameter dot plots that show the average frequencies± SEM of tetramer positive events in the CD4+ population from two animals/group. Data shown are representative of three independent experiments. The data in graphs (B) are expressed as mean values of the absolute number± SEM of IFN-γ releasing CD4+ T cells (left graph) or IFN-γ releasing CD8+T cells (right graph) from two animals/group and three independent experiments. (C) Splenocytes from immunized animals were stimulated with recombinant ESAT6 protein then stained and analyzed by FACS as described in Fig 8. Results are expressed as two-parameter dot plots to show the average frequencies± SEM of IFN-γ producing cell subsets in CD4+ (upper panel) and CD8+ (lower panel) populations from two animals/group. Data shown are representative of three independent experiments. (D) Each column in graph represents the mean of absolute number± SEM of IFN-γ releasing CD4+ (Left graph) or CD8+T cells (right graph) from two animals/group and three independent experiments. *P<0.05; **P<0.01; ***P<0.001.
Mentions: Experiments using the surrogate antigen OVA clearly demonstrated the efficiency of surface decorated BCG to induce specific immune response in vivo. To validate this approach for Mtb specific proteins, we examined mouse immune response to BCG surface decorated with the early secreted Mtb antigen ESAT6, which is a major immunodominant antigen not expressed in BCG [11] and have been previously shown to induce protective T cell response against virulent Mtb [26]. Data from tetramer staining with PE-conjugated I-Ab-ESAT61-20 tetramer (Fig 9A) show that splenocytes from BCG-Avi-ESAT6 immunized mice contained a substantial ESAT6 specific CD4+ T cell subset (0.187±0.032% tetramer positive events) relative to the background value (0.052±0.004%) obtained from splenocytes from wild-type BCG immunized mice. This is also shown in the absolute number of ESAT6 specific CD4+ T cell numbers (Fig 9B). On the other hand, ICS showed significant expansion of IFN-γ producing CD4+ T cells in response to ESAT6 antigen in splenocytes isolated from BCG-Avi-ESAT6 (0.156±0.067%) compared to BCG WT (0.028±0.006%) vaccinated animals (Fig 9C top panel and Fig 9D top graph). Furthermore, the frequencies of IFN-γ producing CD8+ T cells in BCG-Avi-ESAT6 immunized mice were significantly greater than in mice immunized with wild-type BCG (Fig 9C bottom panel and Fig 9D bottom graph). Mice immunized with BCG coated with Avi-ESAT6 were able to induce 0.705±0.065% ESAT6 specific IFN-γ producing CD8+ T cells compared to 0.024±0.006% in BCG WT immunized mice. Taken together, these data showed that BCG surface coated with Mtb specific protein, ESAT6, was able to successfully induce specific ESAT6 immune response in vivo and improve immunogenicity of BCG.

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