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

Abstract\ud \ud \ud \ud Background\ud \ud 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.\ud \ud \ud \ud Methods\ud \ud 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.\ud \ud \ud \ud Results\ud \ud 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.\ud \ud \ud \ud Conclusion\ud \ud 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.We thank Dr. Carlos Rodrigo ZárateBladés for helpful suggestions during the course of the studies. We also thank Mrs. Izaíra T. Brandão and Mrs. Ana P. Masson for technical assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Nacional de DST/AIDS do Ministério da Saúde and Conselho Nacional de Pesquisa (CNPq).We thank Dr. Carlos Rodrigo Zárate-Bladés for helpful suggestions during the course of the studies. We also thank Mrs. Izaíra T. Brandão and Mrs. Ana P. Masson for technical assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Nacional de DST/AIDS do Ministério da Saúde and Conselho Nacional de Pesquisa (CNPq)

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells-2

Timulated 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. < 0.05; < 0.01; < 0.001, in relation to non-stimulated Mφ. #< 0.05; ##< 0.01; ###< 0.001, in relation to non-stimulated DC. (C) Intracellular growth of 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 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). , when compared to CFU numbers recovered on days 0 and 7 postinfection.<p><b>Copyright information:</b></p><p>Taken from "A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells"</p><p>http://www.gvt-journal.com/content/6/1/3</p><p>Genetic Vaccines and Therapy 2008;6():3-3.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2267464.</p><p></p

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells-4

DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.<p><b>Copyright information:</b></p><p>Taken from "A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells"</p><p>http://www.gvt-journal.com/content/6/1/3</p><p>Genetic Vaccines and Therapy 2008;6():3-3.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2267464.</p><p></p

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells-0

DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.<p><b>Copyright information:</b></p><p>Taken from "A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells"</p><p>http://www.gvt-journal.com/content/6/1/3</p><p>Genetic Vaccines and Therapy 2008;6():3-3.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2267464.</p><p></p

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells-1

Indicated by white arrows. (B) Differential capacity of macrophages and DCs to uptake DNA vaccine. Cells were stimulated for 1 h with Alexa Fluor 488-labeled DNA-HSP65 and analysed by flow cytometry. These results are representative of three independent experiments. Black line: stimulated cells; dotted line: non-stimulated cells.<p><b>Copyright information:</b></p><p>Taken from "A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells"</p><p>http://www.gvt-journal.com/content/6/1/3</p><p>Genetic Vaccines and Therapy 2008;6():3-3.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2267464.</p><p></p