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)

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis

Abstract\ud \ud \ud \ud Background\ud \ud Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown.\ud \ud \ud \ud Methods\ud \ud In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge.\ud \ud \ud \ud Results\ud \ud \ud In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice.\ud \ud \ud \ud Conclusions\ud \ud These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.We are thankful to Ana Paula Masson and Izaíra T Brandão for providing the DNA vaccine and recombinant protein. This study was supported by a FAPESP fellowship (05/030873) to LPA.We are thankful to Ana Paula Masson and Izaíra T Brandão for providing the DNA vaccine and recombinant protein. This study was supported by a FAPESP fellowship (05/03087-3) to LPA

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

<p>Abstract</p> <p>Background</p> <p>The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally.</p> <p>Results</p> <p>We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 μg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-γ and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 μg).</p> <p>Conclusion</p> <p>Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.</p

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Abstract Background mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.</p

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.This work was supported by FAPESP (06/03987-7) grant. We thank Ana P.\ud Masson and Izaíra T. Brandão for technical support

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