Célio Lopes Silva

Protection against tuberculosis by a plasmid DNA vaccine

Past attempts to use fractions of mycobacteria as an alternative to BCG have given disappointing results. The availability of cloned genes and suitable vectors has now opened a new avenue in which individual mycobacterial protein antigens are synthesised within transfected mammalian cells. In an ex vivo transfection approach with a retroviral vector we found that even a single antigen (hsp65) could evoke strong protection when expressed as a transgene and that expression of protection was largely a function of antigen specific cytotoxic T cells. We now find that intramuscular injection of plasmid DNA expressing the antigen from either a viral or a murine promoter can also give protection equivalent to Bacillus Calmette-Guérin (BCG). Plasmids expressing some other mycobacterial antigens, hsp70, 36 kDa and 6 kDa, are also effective, suggesting that this approach may lead to a new vaccine.

Towards a DNA vaccine against tuberculosis

Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp65) in adult Balb/c mice resulted in substantial cell-mediated protection against challenge with M. tuberculosis. CD4 and CD8 T cells cloned from spleens of such immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. Three modes of expressing the gene have been tested: (1) expression from a retroviral vector (pZIPNeoSV) in implanted J774 tumour cells, (2) expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and (3) in a preliminary experiment, from CMV immediate-early and hydroxymethylglutaryl Co-A reductase promoters injected as plasmid DNA into muscle.

Blockade of Endogenous Leukotrienes Exacerbates Pulmonary Histoplasmosis

ABSTRACT Leukotrienes are classical mediators of inflammatory response. New aspects of leukotriene function have recently been described. We examine here the previously unreported role that leukotrienes play in the regulation of cytokines in a murine model of histoplasmosis. We demonstrate that administration of MK 886, a leukotriene synthesis inhibitor, caused Histoplasma capsulatum-infected mice to die by the day 15 of infection, whereas the correlating death rate in untreated infected mice was 0%. Treating infected animals with MK 886 inhibited leukotriene synthesis but increased leukocyte recruitment to the lungs. Subsequent to this phenomenon, levels of tumor necrosis factor alpha, interleukin-1 (IL-1), IL-6, and KC chemoattractant cytokines and fungi in the lung parenchyma increased, as did inflammatory response. In contrast, IL-2, IL-5, IL-12, and gamma interferon cytokine levels actually decreased. Thus, murine response to pulmonary histoplasmosis may be leukotriene modulated. This finding may enable us to alter the course of the immune response and inflammation caused by histoplasmosis. The data from the present study suggest an important new strategy for immunologic or drug intervention in human patients.

Identification and Characterization of Murine Cytotoxic T Cells That Kill Mycobacterium tuberculosis

ABSTRACT As we seek to develop and evaluate new vaccines against tuberculosis, it is desirable that we understand the mechanisms of protective immunity in our models. Adoptive transfer of protection with hsp65-specific T-cell clones from infected or vaccinated mice into naı̈ve mice had indicated that cytotoxic T cells can make a major contribution to protection. We characterized 28 CD4+CD8− and 28 CD4− CD8+hsp65-specific T-cell clones derived from infected or vaccinated mice. Half of the CD4+ CD8− and 64% of the CD4− CD8+ clones were cytotoxic. Cytotoxicity was associated with high expression of CD44 and gamma interferon production. Most (86%) of the cytotoxic CD4+CD8− clones lysed target cells via the Fas-FasL pathway, and most (83%) of the cytotoxic CD4− CD8+clones lysed target cells via cytotoxic granules. Only the clones using the granule-mediated pathway caused substantial loss of viability of virulent Mycobacterium tuberculosis during lysis of infected macrophages, and the degree of killing closely correlated with the availability of granule marker enzyme activity. Granule-mediated cytotoxicity thus may have a key role in protection against tuberculosis by delivering mycobactericidal granule contents.

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

BackgroundThe 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.ResultsWe 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).ConclusionOur 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.

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

BackgroundmRNAs 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.ResultsWe 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).ConclusionsTaken 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.

Role of Trehalose Dimycolate in Recruitment of Cells and Modulation of Production of Cytokines and NO in Tuberculosis

ABSTRACT Mice treated with viable Mycobacterium tuberculosis with no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidated M. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-dl-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-12, IL-10, gamma interferon (IFN-γ), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-γ, IL-6, TNF-α, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon-γ recovery and reduction of lung injury

A DNA vaccine based on the heat‐shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis‐infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65‐treated mice and infected pCDNA3‐treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon‐γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor‐α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65‐treated mice were able to produce significant levels of interferon‐γ and to restrict the growth of bacilli.

Leukotrienes are involved in leukocyte recruitment induced by live Histoplasma capsulatum or by the β‐glucan present in their cell wall

The inflammatory cell influx towards the peritoneal cavity in mice inoculated i.p. with live or dead Histoplasma capsulatum or with its subcellular preparations was studied. We also evaluated the effects of dexamethasone (Dexa) or MK886, an inhibitor of leukotriene (LT) biosynthesis, on the recruitment of leukocytes. Live yeast form of fungus (LYH) induced an increase in neutrophils (NE) which was highest 4 to 24 h after inoculation. Mononuclear cell (MN) migration beginning at 24 h with a gradual increase over 48 and 168 h, and an eosinophil (EO) recruitment occurs between 24 and 48 h. NE and EO recruitment induced by dead mycelial form of fungus (DMH) was greater than that observed for dead yeast form of fungus (DYH). A similar leukocyte migration pattern was seen after i.p. injection of the alkali‐insoluble fraction (F1) from DYH (F1Y) and F1 from DMH (F1M) this being more active than former. The difference in concentration of β‐glucan in DYH and DMH could explain the different inflammatory capacity exhibited by the two forms of H. capsulatum. LT seems to be the principal mediator of leukocyte migration in response to LYH, DYH or DMH or to β‐glucan. However, other mediators appear to contribute to NE and EO migration since the treatment with Dexa was more effective in inhibiting cell migration than MK886. Complement dependent leukocyte migration may participate in this recruitment. Treatment with MK886 completely abolished MN cell migration, indicating its dependence on the presence of LT.

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 µmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.