Vania L. D. Bonato

Immunotherapy with plasmid DNA encoding mycobacterial hsp65 in association with chemotherapy is a more rapid and efficient form of treatment for tuberculosis in mice

Tuberculosis (TB) remains a threat for public health, killing around 3 million people a year. Despite the fact that most cases can be cured with antibiotics, the treatment is long and patients relapse if chemotherapy is not continued for at least 6 months. Thus, a better characterization of the working principles of the immune system in TB and identification of new immunotherapeutic products for the development of shorter regimens of treatment are essential to achieve an effective management of this disease. In the present work, we demonstrate that immunotherapy with a plasmid DNA encoding the Mycobacterium leprae 65 kDa heat-shock protein (hsp65) in order to boost the efficiency of the immune system, is a valuable adjunct to antibacterial chemotherapy to shorten the duration of treatment, improve the treatment of latent TB infection and be effective against multidrug-resistant bacilli (MDR-TB). We also showed that the use of DNA-hsp65 alone or in combination with other drugs influence the pathway of the immune response or other types of inflammatory responses and should augment our ability to alter the course of immune response/inflammation as needed, evidencing an important target for immunization or drug intervention.

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.

Comparison of different delivery systems of vaccination for the induction of protection against tuberculosis in mice

The way to deliver antigens and cellular requirements for long-lasting protection against tuberculosis are not known. Immunizations with mycobacterial 65 kDa heat shock protein (hsp65) expressed from J774-hsp65 cells (antigen-presenting cells that endogenously produce hsp65 antigen) or from plasmid DNA, or with the protein entrapped in cationic liposomes, can each give protective immunity similar to that obtained from live Bacillus Calmette Guérin (BCG), whereas injecting the protein in Freunds incomplete adjuvant (FIA) has minimal effect. Protective procedures elicited high frequencies of antigen-reactive alphabeta T cells with CD4+/CD8- and CD8+/CD4- phenotypes. Protection correlated with the abundance of hsp65-dependent cytotoxic CD8+/CD4-/CD44hi cells. The frequency of these cells and the level of protection declined during 8 months after J774-hsp65 or liposome-mediated immunization with hsp65 protein but were sustained or steadily increased over this period after hsp65-DNA or BCG immunizations. IFN-gamma predominated over IL-4 among the hsp65-reactive CD8+/CD4- and CD4+/CD8- populations after J774-hsp65-, hsp65-liposome-, and hsp65-DNA-mediated immunizations, but similar levels of these cytokines prevailed after BCG vaccination.

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime–boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime–boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine.

Recent advances in DNA vaccines for autoimmune diseases

Vaccination is one of the most powerful health tools available owing to its ability to confer protection against various diseases. The long-term impact of such protection in terms of public-health savings is nearly incalculable and becomes even more evident when considering if the vaccination concept is extended to the therapeutic potential of a given molecule. In this sense, DNA vaccines are especially important tools with enormous potential owing to the molecular precision that they offer. The properties of the plasmid DNA molecule in terms of stability, cost–effectiveness and lack of cold-chain requirement are additional advantages over traditional vaccines and therapeutics. We focus on the current knowledge of autoimmune mechanisms, engineering of DNA vaccines and attempts that have already been made in order to intervene in autoimmune processes. Our experience with a genetic vaccine containing the heat-shock protein gene (hsp65) from mycobacteria is also described.

IFN-γ-mediated efficacy of allergen-free immunotherapy using mycobacterial antigens and CpG-ODN.

Epidemiological and experimental evidence supports the notion that microbial infections that are known to induce Th1‐type immune responses can suppress Th2 immune responses, which are characteristics of allergic disorders. However, live microbial immunization might not be feasible for human immunotherapy. Here, we evaluated whether induction of Th1 immunity by the immunostimulatory sequences of CpG‐oligodeoxynucleotides (CpG‐ODN), with or without culture filtrate proteins (CFP), from Mycobacterium tuberculosis would suppress ongoing allergic lung disease. Presensitized and ovalbumin (OVA)‐challenged mice were treated subcutaneously with CpG, or CpG in combination with CFP (CpG/CFP). After 15 days of treatment, airway inflammation and specific T‐ and B‐cell responses were determined. Cell transfer experiments were also performed. CpG treatment attenuated airway allergic disease; however, the combination CpG/CFP treatment was significantly more effective in decreasing airway hyperresponsiveness, eosinophilia and Th2 response. When an additional intranasal dose of CFP was given, allergy was even more attenuated. The CpG/CFP therapy also reduced allergen‐specific IgG1 and IgE antibodies and increased IgG2a. Transfer of spleen cells from mice immunized with CpG/CFP also reduced allergic lung inflammation. CpG/CFP treatment induced CFP‐specific production of IFN‐γ and IL‐10 by spleen cells and increased production of IFN‐γ in response to OVA. The essential role of IFN‐γ for the therapeutic effect of CpG/CFP was evidenced in IFN‐γ knockout mice. These results show that CpG/CFP treatment reverses established Th2 allergic responses by an IFN‐γ‐dependent mechanism that seems to act both locally in the lung and systemically to decrease allergen‐specific Th2 responses.

Oral administration of Saccharomyces cerevisiae UFMG A-905 prevents allergic asthma in mice

The prevalence of asthma has increased in communities that adopt a Western lifestyle and become more urbanized. Probiotics may be effective in the prevention of allergic diseases, such as asthma. The aim of the current study was to examine the effects of Saccharomyces cerevisiae UFMG A‐905 in an allergic model of asthma.