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A new recombinant BCG vaccine induces specific Th17 and Th1 effector cells with higher protective efficacy against tuberculosis.

da Costa AC, Costa-Júnior Ade O, de Oliveira FM, Nogueira SV, Rosa JD, Resende DP, Kipnis A, Junqueira-Kipnis AP - PLoS ONE (2014)

Bottom Line: Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG.Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load.In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunopatologia das Doenças Infecciosas, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.

ABSTRACT
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.

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Immunogenicity of rBCG-CMX in BALB/c mice.(A) Experimental time line. BALB/c mice were immunized with rBCG-CMX or BCG Moreau. Thirty days later, 6 mice per group were euthanized for evaluation of vaccine-induced immunogenicity. Ninety days after immunization, mice were intravenously (i.v.) challenged with 105 CFU of H37Rv. Forty-five days after i.v. challenge, the lung bacterial load (CFU) and lesions (H&E) were assessed. (B–E) Specific cellular immune responses induced with rCMX stimulation exvivo. Spleen (B and D) and lung (C and E) cell suspensions from vaccinated and unvaccinated (Control) mice were stimulated with rCMX. Cells positive for both CD4 and IFN-γ (B and C) or CD4 and IL-17 (D and E) were determined by flow cytometry. Lymphocytes were selected based on size and granularity. Flow cytometry gates were set to analyze CD4+ T cells, and then the fluorescence of antibodies detecting IFN-γ+ or IL-17+ cells was recorded. These data are representative of two independent experiments (N = 6, *p<0.05).
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pone-0112848-g004: Immunogenicity of rBCG-CMX in BALB/c mice.(A) Experimental time line. BALB/c mice were immunized with rBCG-CMX or BCG Moreau. Thirty days later, 6 mice per group were euthanized for evaluation of vaccine-induced immunogenicity. Ninety days after immunization, mice were intravenously (i.v.) challenged with 105 CFU of H37Rv. Forty-five days after i.v. challenge, the lung bacterial load (CFU) and lesions (H&E) were assessed. (B–E) Specific cellular immune responses induced with rCMX stimulation exvivo. Spleen (B and D) and lung (C and E) cell suspensions from vaccinated and unvaccinated (Control) mice were stimulated with rCMX. Cells positive for both CD4 and IFN-γ (B and C) or CD4 and IL-17 (D and E) were determined by flow cytometry. Lymphocytes were selected based on size and granularity. Flow cytometry gates were set to analyze CD4+ T cells, and then the fluorescence of antibodies detecting IFN-γ+ or IL-17+ cells was recorded. These data are representative of two independent experiments (N = 6, *p<0.05).

Mentions: Since rBCG-CMX was stable invivo and phagocytosis of it induced macrophage activation (as measured by NO production), we questioned whether this vaccine would be able to induce a specific response to CMX and/or to the recombinant antigens alone (Fig. 4A). Immunization with rBCG-CMX vaccine induced higher numbers of CD4+ T lymphocytes positive for IFN-γ specific for CMX in cells from the spleen and lungs of BALB/c immunized mice 30 days after vaccination than did immunization with BCG Moreau (Figs. 4B and C, p<0.05; Th1 representative dot plots in Figure S2A). Similarly, rBCG-CMX induced higher levels of specific Th17 cells, an important group of cells for protection from Mtb and development of memory, in the cells from the spleen and lungs of immunized mice (Figs. 4D and E, p<0.05; Th17 representative dot plots in Fig. S2B).


A new recombinant BCG vaccine induces specific Th17 and Th1 effector cells with higher protective efficacy against tuberculosis.

da Costa AC, Costa-Júnior Ade O, de Oliveira FM, Nogueira SV, Rosa JD, Resende DP, Kipnis A, Junqueira-Kipnis AP - PLoS ONE (2014)

Immunogenicity of rBCG-CMX in BALB/c mice.(A) Experimental time line. BALB/c mice were immunized with rBCG-CMX or BCG Moreau. Thirty days later, 6 mice per group were euthanized for evaluation of vaccine-induced immunogenicity. Ninety days after immunization, mice were intravenously (i.v.) challenged with 105 CFU of H37Rv. Forty-five days after i.v. challenge, the lung bacterial load (CFU) and lesions (H&E) were assessed. (B–E) Specific cellular immune responses induced with rCMX stimulation exvivo. Spleen (B and D) and lung (C and E) cell suspensions from vaccinated and unvaccinated (Control) mice were stimulated with rCMX. Cells positive for both CD4 and IFN-γ (B and C) or CD4 and IL-17 (D and E) were determined by flow cytometry. Lymphocytes were selected based on size and granularity. Flow cytometry gates were set to analyze CD4+ T cells, and then the fluorescence of antibodies detecting IFN-γ+ or IL-17+ cells was recorded. These data are representative of two independent experiments (N = 6, *p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232451&req=5

pone-0112848-g004: Immunogenicity of rBCG-CMX in BALB/c mice.(A) Experimental time line. BALB/c mice were immunized with rBCG-CMX or BCG Moreau. Thirty days later, 6 mice per group were euthanized for evaluation of vaccine-induced immunogenicity. Ninety days after immunization, mice were intravenously (i.v.) challenged with 105 CFU of H37Rv. Forty-five days after i.v. challenge, the lung bacterial load (CFU) and lesions (H&E) were assessed. (B–E) Specific cellular immune responses induced with rCMX stimulation exvivo. Spleen (B and D) and lung (C and E) cell suspensions from vaccinated and unvaccinated (Control) mice were stimulated with rCMX. Cells positive for both CD4 and IFN-γ (B and C) or CD4 and IL-17 (D and E) were determined by flow cytometry. Lymphocytes were selected based on size and granularity. Flow cytometry gates were set to analyze CD4+ T cells, and then the fluorescence of antibodies detecting IFN-γ+ or IL-17+ cells was recorded. These data are representative of two independent experiments (N = 6, *p<0.05).
Mentions: Since rBCG-CMX was stable invivo and phagocytosis of it induced macrophage activation (as measured by NO production), we questioned whether this vaccine would be able to induce a specific response to CMX and/or to the recombinant antigens alone (Fig. 4A). Immunization with rBCG-CMX vaccine induced higher numbers of CD4+ T lymphocytes positive for IFN-γ specific for CMX in cells from the spleen and lungs of BALB/c immunized mice 30 days after vaccination than did immunization with BCG Moreau (Figs. 4B and C, p<0.05; Th1 representative dot plots in Figure S2A). Similarly, rBCG-CMX induced higher levels of specific Th17 cells, an important group of cells for protection from Mtb and development of memory, in the cells from the spleen and lungs of immunized mice (Figs. 4D and E, p<0.05; Th17 representative dot plots in Fig. S2B).

Bottom Line: Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG.Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load.In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunopatologia das Doenças Infecciosas, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.

ABSTRACT
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.

Show MeSH
Related in: MedlinePlus