Vânia Luiza Deperon Bonato

Characterization of the memory/activated T cells that mediate the long-lived host response against tuberculosis after bacillus Calmette–Guérin or DNA vaccination

The memory/activated T cells, which mediate the long‐lived host response against tuberculosis, in mice immunized with either bacillus Calmette–Guérin (BCG) or mycobacterium heat‐shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA‐hsp 65), were characterized. Protection against Mycobacterium tuberculosis challenge by DNA‐hsp 65 vaccination was associated with the presence of lymph node T‐cell populations in which CD8+/CD44hi interferon‐γ (IFN‐γ)‐producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA‐mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN‐γ‐producing T cells. When the cells were separated into CD4+CD8− and CD8+CD4− and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4− and those from DNA‐vaccinated mice were much more protective than those from BCG‐immunized mice. The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Increased levels of interferon-γ primed by culture filtrate proteins antigen and CpG-ODN immunization do not confer significant protection against Mycobacterium tuberculosis infection

The results of various animal model studies of tuberculosis (TB) suggest that culture filtrate proteins (CFPs), which are antigens secreted by Mycobacterium tuberculosis, are largely responsible for improvements in TB vaccines. The great obstacle to developing protein subunit vaccines is that adjuvants are required in order to stimulate relevant protective immune responses. Acting as immune adjuvants, CpG‐oligodeoxynucleotides (CpG‐ODNs) promote the activation of Th1 cells and of pro‐inflammatory cytokines. To evaluate the adjuvant role of CpG‐ODNs in conferring enhanced immunogenic capacity and protection against M. tuberculosis, we immunized mice with CFP antigen combined with synthetic CpG‐ODNs (CFP/CpG) or with incomplete Freunds adjuvant (IFA) (CFP/IFA). Immunization with CFP/CpG induced a T helper 1 (Th1)‐biased response accompanied by a higher immunoglobulin G2a (IgG2a) antibody/IgG1 antibody ratio, elevated production of interferon‐γ (IFN‐γ) by spleen cells and in lungs. However, CFP/IFA‐immunized mice presented higher levels of IgG1 antibodies, as well as increased production of IFN‐γ, interleukin (IL)‐5, and IL‐10 by spleen cells, together with lower levels of IFN‐γ in the lungs. Despite the stronger Th1 response seen in both groups, believed to be necessary for protection against TB, only mice immunized with CFP/IFA were protected after M. tuberculosis infection. Lung histology revealed that lung parenchyma were better preserved in CFP/IFA‐immunized mice, which also presented intense lymphocyte recruitment to the lesion, whereas CFP/CpG‐immunized mice presented severe pulmonary injury accompanied by necrosis. Based on the data presented, we discuss the widely accepted paradigm that high levels of IFN‐γ are directly correlated with protection against experimental TB.

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.

Histoplasma capsulatum Inhibits Apoptosis and Mac‐1 Expression in Leucocytes

Histoplasma capsulatum is a fungus found intracellularly in neutrophils and peripheral blood mononuclear cells (PBMCs), suggesting that it is capable of evading damage and survives inside these cells. In this study, we report that neutrophils from H. capsulatum ‐infected mice, and human neutrophils and mononuclear cells exposed to H. capsulatum presented less apoptosis than those from noninfected animals or cells exposed to medium only. Moreover, cells harvested from infected animals are resistant to apoptosis induced by dexamethasone – a proapoptotic stimulant. We also show that neutrophils harvested from infected mice and PBMCs from humans exposed to the fungus had a greatly decreased Mac‐1 expression. We conclude that H. capsulatum induces an antiapoptotic state on leucocytes, which correlates with decreased cell‐surface Mac‐1 expression. These facts may represent an escape mechanism for the fungus by delaying cell death and allowing the fungus to survive inside leucocytes.

Host genetic background affects regulatory T-cell activity that influences the magnitude of cellular immune response against Mycobacterium tuberculosis.

Using two mouse strains with different abilities to generate interferon (IFN)‐γ production after Mycobacterium tuberculosis infection, we tested the hypothesis that the frequency and activity of regulatory T (Treg) cells are influenced by genetic background. Our results demonstrated that the suppressive activity of spleen Treg cells from infected or uninfected BALB/c mice was enhanced, inhibiting IFN‐γ and interleukin (IL)‐2 production. Infected C57BL/6 mice exhibited a decrease in the frequency of lung Treg cells and an increased ratio CD4+:CD4+Foxp3+ cells compared with infected BALB/c mice and uninfected C57BL/6 mice. Moreover, infected C57BL/6 mice also had a decrease in the immunosuppressive capacity of spleen Treg cells, higher lung IFN‐γ and IL‐17 production, and restricted the infection better than BALB/c mice. Adoptive transfer of BALB/c Treg cells into BALB/c mice induced an increase in bacterial colony‐forming unit (CFU) counts. Furthermore, BALB/c mice treated with anti‐CD25 antibody exhibited lung CFU counts significantly lower than mice treated with irrelevant antibody. Our results show that in BALB/c mice, the Treg cells have a stronger influence than that in C57BL/6 mice. These data suggest that BALB/c and C57BL/6 mice may use some different mechanisms to control M. tuberculosis infection. Therefore, the role of Treg cells should be explored during the development of immune modulators, both from the perspective of the pathogen and the host.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

ABSTRACT Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-γ) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-γ-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-γ and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-γ and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR‐TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood.

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4+/CD4+ Foxp3+ cells

CD4+ Foxp3+ regulatory T cells inhibit the production of interferon‐γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat‐shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4+ Foxp3+ cells compared with non‐immunized mice. Heterologous immunization using bacillus Calmette–Guérin (BCG) to prime and DNA‐hsp 65 to boost (BCG/DNA‐hsp 65) or BCG to prime and culture filtrate proteins (CFP)‐CpG to boost (BCG/CFP‐CpG) induced a significantly higher ratio of spleen CD4+/CD4+ Foxp3+ cells compared with non‐immunized mice. In addition, the protection conferred by either the BCG/DNA‐hsp 65 or the BCG/CFP‐CpG vaccines was significant compared with the DNA‐hsp 65 vaccine. Despite the higher ratio of spleen CD4+/CD4+ Foxp3+ cells found in BCG/DNA‐hsp 65‐immunized or BCG/CFP‐CpG‐immunized mice, the lungs of both groups of mice were better preserved than those of DNA‐hsp 65‐immunized mice. These results confirm the protective efficacy of BCG/DNA‐hsp 65 and BCG/CFP‐CpG heterologous prime‐boost vaccines and the DNA‐hsp 65 homologous vaccine. Additionally, the prime‐boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4+ and regulatory (CD4+ Foxp3+) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway.

Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non‐infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60–65 kDa heat shock protein (Hsp) family is endowed with anti‐inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders.