Ana Flávia Gembre

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.

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.

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.

CD11c(+) CD103(+) cells of Mycobacterium tuberculosis-infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon-γ- or interleukin-17-producing CD4(+) cells.

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4+ populations generated after M. tuberculosis infection. Thirty days post‐infection, C57BL/6 mice, which generate a stronger interferon‐γ (IFN‐γ)‐mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c+ CD11b− CD103+ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c+ CD11b+ CD103− cells. CD11c+ CD11b− CD103+ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN‐γ‐producing or interleukin‐17 (IL‐17)‐producing CD4+ cells. Moreover, CD4+ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN‐γ‐producing or IL‐17‐producing CD4+ cells is dependent on CD11c+ CD11b− CD103+ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.

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.

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

BackgroundAlthough B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown.MethodsIn this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge.ResultsIn 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.ConclusionsThese 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.

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.

Attenuation of experimental asthma by mycobacterial protein combined with CpG requires a TLR9‐dependent IFN‐γ‐CCR2 signalling circuit

Allergic asthma is a chronic pulmonary disease characterized by a Th2 inflammatory response. The modulation of a Th2 immune response based on immune deviation to a Th1 pattern or induction and migration of regulatory T cells to the lungs constitutes one of the major therapeutic approaches that is being investigated for the treatment of allergic asthma. The potentials of Mycobacterium leprae 65‐kD heat‐shock protein or Toll‐like receptor 9 ligand (CpG oligodeoxynucleotides) as immune modulators for the treatment of airway allergic disease have been studied individually.

Genetic background affects the expansion of macrophage subsets in the lungs of Mycobacterium tuberculosis-infected hosts

M1 macrophages are more effective in the induction of the inflammatory response and clearance of Mycobacterium tuberculosis than M2 macrophages. Infected C57BL/6 mice generate a stronger cellular immune response compared with BALB/c mice. We hypothesized that infected C57BL/6 mice would exhibit a higher frequency and function of M1 macrophages than infected BALB/c mice. Our findings show a higher ratio of macrophages to M2 macrophages in the lungs of chronically infected C57BL/6 mice compared with BALB/c mice. However, there was no difference in the functional ability of M1 and M2 macrophages for the two strains in vitro. In vivo, a deleterious role for M2 macrophages was confirmed by M2 cell transfer, which rendered the infected C57BL/6, but not the BALB/c mice, more susceptible and resulted in mild lung inflammation compared with C57BL/6 mice that did not undergo cell transfer. M1 cell transfer induced a higher inflammatory response, although not protective, in infected BALB/c mice compared with their counterparts that did not undergo cell transfer. These findings demonstrate that an inflammation mediated by M1 macrophages may not induce bacterial tolerance because protection depends on the host genetic background, which drives the magnitude of the inflammatory response against M. tuberculosis in the pulmonary microenvironment. The contribution of our findings is that although M1 macrophage is an effector leucocyte with microbicidal machinery, its dominant role depends on the balance of M1 and M2 subsets, which is driven by the host genetic background.