Marina Oliveira e Paula

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.

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.

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.

Experimental tuberculosis: Designing a better model to test vaccines against tuberculosis☆

Experimental models of infection are good tools for establishing immunological parameters that have an effect on the host-pathogen relationship and also for designing new vaccines and immune therapies. In this work, we evaluated the evolution of experimental tuberculosis in mice infected with increasing bacterial doses or via distinct routes. We showed that mice infected with low bacterial doses by the intratracheal route were able to develop a progressive infection that was proportional to the inoculum size. In the initial phase of disease, mice developed a specific Th1-driven immune response independent of inoculum concentration. However, in the late phase, mice infected with higher concentrations exhibited a mixed Th1/Th2 response, while mice infected with lower concentrations sustained the Th1 pattern. Significant IL-10 concentrations and a more preeminent T regulatory cell recruitment were also detected at 70 days post-infection with high bacterial doses. These results suggest that mice infected with higher concentrations of bacilli developed an immune response similar to the pattern described for human tuberculosis wherein patients with progressive tuberculosis exhibit a down modulation of IFN-gamma production accompanied by increased levels of IL-4. Thus, these data indicate that the experimental model is important in evaluating the protective efficacy of new vaccines and therapies against tuberculosis.

Requirement of MyD88 and Fas pathways for the efficacy of allergen-free immunotherapy

We have shown that mycobacterial antigens and CpG oligodeoxynucleotides downmodulate airway allergic inflammation by mechanisms dependent on T‐cell activation. Here, we investigated the participation of the innate response, particularly the role of MyD88 adaptor, and Fas molecules in the effectiveness of DNA‐HSP65 or CpG/culture filtrated proteins (CFP) immunotherapy.

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients

ABSTRACT Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.