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A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells.

Franco LH, Wowk PF, Silva CL, Trombone AP, Coelho-Castelo AA, Oliver C, Jamur MC, Moretto EL, Bonato VL - Genet Vaccines Ther (2008)

Bottom Line: As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans.These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Av, Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brasil. luishenrique@cpt.fmrp.usp.br

ABSTRACT

Background: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.

Methods: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.

Results: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.

Conclusion: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Activation of the innate immune response mediated by DNA-HSP65. (A) Expression of costimulatory molecules and HLA-DR on the surface of macrophages (Mφ) and DC stimulated with DNA vaccine. Cells were stimulated with DNA vaccine, DNA vector or LPS (positive control). After 48 h stimulation, the expression of surface molecules was evaluated by flow cytometry. Each column represents the mean percentage of Mφ or DC positive for CD80, CD86 or HLA-DR, or DC positive for CD83 ± SEM. Cells were obtained from 11 cultures of Mφ and 7–9 cultures of DC from different healthy individuals. (B) Mφ and DC were incubated for 48 h with DNA vaccine, DNA vector or LPS and the production of TNF-alpha, IL-6, IL-10 and IL-12p40 was evaluated. Each column represents the mean ± SEM of cytokine production detected in 6–8 Mφ cultures or 7–10 DC cultures obtained from healthy donors. *p < 0.05; **p < 0.01; ***p < 0.001, in relation to non-stimulated Mφ. #p < 0.05; ##p < 0.01; ###p < 0.001, in relation to non-stimulated DC. (C) Intracellular growth of M. tuberculosis in Mφ or DCs stimulated with DNA-HSP65. Mφ and DCs were stimulated with DNA vaccine or DNA vector (both at 20 μg/mL) for 48 h and infected with M. tuberculosis at MOI = 1. CFU numbers were determined at 4 h (day 0) and 7 days (day 7) after infection. Results represent the mean ± SEM of five experiments (for DCs) or three experiments (for Mφ). * p < 0,05, when compared to CFU numbers recovered on days 0 and 7 postinfection.
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Figure 3: Activation of the innate immune response mediated by DNA-HSP65. (A) Expression of costimulatory molecules and HLA-DR on the surface of macrophages (Mφ) and DC stimulated with DNA vaccine. Cells were stimulated with DNA vaccine, DNA vector or LPS (positive control). After 48 h stimulation, the expression of surface molecules was evaluated by flow cytometry. Each column represents the mean percentage of Mφ or DC positive for CD80, CD86 or HLA-DR, or DC positive for CD83 ± SEM. Cells were obtained from 11 cultures of Mφ and 7–9 cultures of DC from different healthy individuals. (B) Mφ and DC were incubated for 48 h with DNA vaccine, DNA vector or LPS and the production of TNF-alpha, IL-6, IL-10 and IL-12p40 was evaluated. Each column represents the mean ± SEM of cytokine production detected in 6–8 Mφ cultures or 7–10 DC cultures obtained from healthy donors. *p < 0.05; **p < 0.01; ***p < 0.001, in relation to non-stimulated Mφ. #p < 0.05; ##p < 0.01; ###p < 0.001, in relation to non-stimulated DC. (C) Intracellular growth of M. tuberculosis in Mφ or DCs stimulated with DNA-HSP65. Mφ and DCs were stimulated with DNA vaccine or DNA vector (both at 20 μg/mL) for 48 h and infected with M. tuberculosis at MOI = 1. CFU numbers were determined at 4 h (day 0) and 7 days (day 7) after infection. Results represent the mean ± SEM of five experiments (for DCs) or three experiments (for Mφ). * p < 0,05, when compared to CFU numbers recovered on days 0 and 7 postinfection.

Mentions: In order to study the activation of innate immune response mediated by DNA-HSP65, human macrophages and DCs stimulated with the DNA vaccine were evaluated regarding the cytokine production, expression of surface markers and microbicidal activity. In relation to cellular phenotype, we did not observe any variation in the number of DNA vaccine-stimulated macrophages expressing HLA-DR, CD80 or CD86 molecules (Figure 3A) or changes in the median fluorescence intensity (data not shown). Conversely, the stimulation of DCs with DNA vaccine resulted in an up-regulation of CD80, CD86 and CD83 (a maturation marker) expression (Figure 3A). After stimulation with DNA vaccine or DNA vector, no difference was seen in the number of DCs expressing HLA-DR. In all experiments LPS was used as positive control of cellular activation.


A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells.

Franco LH, Wowk PF, Silva CL, Trombone AP, Coelho-Castelo AA, Oliver C, Jamur MC, Moretto EL, Bonato VL - Genet Vaccines Ther (2008)

Activation of the innate immune response mediated by DNA-HSP65. (A) Expression of costimulatory molecules and HLA-DR on the surface of macrophages (Mφ) and DC stimulated with DNA vaccine. Cells were stimulated with DNA vaccine, DNA vector or LPS (positive control). After 48 h stimulation, the expression of surface molecules was evaluated by flow cytometry. Each column represents the mean percentage of Mφ or DC positive for CD80, CD86 or HLA-DR, or DC positive for CD83 ± SEM. Cells were obtained from 11 cultures of Mφ and 7–9 cultures of DC from different healthy individuals. (B) Mφ and DC were incubated for 48 h with DNA vaccine, DNA vector or LPS and the production of TNF-alpha, IL-6, IL-10 and IL-12p40 was evaluated. Each column represents the mean ± SEM of cytokine production detected in 6–8 Mφ cultures or 7–10 DC cultures obtained from healthy donors. *p < 0.05; **p < 0.01; ***p < 0.001, in relation to non-stimulated Mφ. #p < 0.05; ##p < 0.01; ###p < 0.001, in relation to non-stimulated DC. (C) Intracellular growth of M. tuberculosis in Mφ or DCs stimulated with DNA-HSP65. Mφ and DCs were stimulated with DNA vaccine or DNA vector (both at 20 μg/mL) for 48 h and infected with M. tuberculosis at MOI = 1. CFU numbers were determined at 4 h (day 0) and 7 days (day 7) after infection. Results represent the mean ± SEM of five experiments (for DCs) or three experiments (for Mφ). * p < 0,05, when compared to CFU numbers recovered on days 0 and 7 postinfection.
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Related In: Results  -  Collection

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Figure 3: Activation of the innate immune response mediated by DNA-HSP65. (A) Expression of costimulatory molecules and HLA-DR on the surface of macrophages (Mφ) and DC stimulated with DNA vaccine. Cells were stimulated with DNA vaccine, DNA vector or LPS (positive control). After 48 h stimulation, the expression of surface molecules was evaluated by flow cytometry. Each column represents the mean percentage of Mφ or DC positive for CD80, CD86 or HLA-DR, or DC positive for CD83 ± SEM. Cells were obtained from 11 cultures of Mφ and 7–9 cultures of DC from different healthy individuals. (B) Mφ and DC were incubated for 48 h with DNA vaccine, DNA vector or LPS and the production of TNF-alpha, IL-6, IL-10 and IL-12p40 was evaluated. Each column represents the mean ± SEM of cytokine production detected in 6–8 Mφ cultures or 7–10 DC cultures obtained from healthy donors. *p < 0.05; **p < 0.01; ***p < 0.001, in relation to non-stimulated Mφ. #p < 0.05; ##p < 0.01; ###p < 0.001, in relation to non-stimulated DC. (C) Intracellular growth of M. tuberculosis in Mφ or DCs stimulated with DNA-HSP65. Mφ and DCs were stimulated with DNA vaccine or DNA vector (both at 20 μg/mL) for 48 h and infected with M. tuberculosis at MOI = 1. CFU numbers were determined at 4 h (day 0) and 7 days (day 7) after infection. Results represent the mean ± SEM of five experiments (for DCs) or three experiments (for Mφ). * p < 0,05, when compared to CFU numbers recovered on days 0 and 7 postinfection.
Mentions: In order to study the activation of innate immune response mediated by DNA-HSP65, human macrophages and DCs stimulated with the DNA vaccine were evaluated regarding the cytokine production, expression of surface markers and microbicidal activity. In relation to cellular phenotype, we did not observe any variation in the number of DNA vaccine-stimulated macrophages expressing HLA-DR, CD80 or CD86 molecules (Figure 3A) or changes in the median fluorescence intensity (data not shown). Conversely, the stimulation of DCs with DNA vaccine resulted in an up-regulation of CD80, CD86 and CD83 (a maturation marker) expression (Figure 3A). After stimulation with DNA vaccine or DNA vector, no difference was seen in the number of DCs expressing HLA-DR. In all experiments LPS was used as positive control of cellular activation.

Bottom Line: As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans.These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Av, Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brasil. luishenrique@cpt.fmrp.usp.br

ABSTRACT

Background: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.

Methods: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.

Results: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.

Conclusion: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

No MeSH data available.


Related in: MedlinePlus