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IFN-gamma, IL-12, IL-10 and TNF-alpha in lung homogenates from immunized, infected mice and infected mice after 30 days of the infection. (A) IFN-gamma; (B) IL-12; (C) IL-10 and (D) TNF-alpha. (E) Correlation between CFU numbers and IFN-gamma production (F) Correlation between IFN-gamma and IL-10 production. Groups of 7 mice were immunized according table I and 15 days after the last immunization, they were challenged with H37Rv. After 30 days of infection, the lungs were removed and the cytokine production in lungs homogenates was analyzed. Bars represent the mean ± standard deviation. (A) ◆ BCGsc, BCGin, DNA-HSP65, BCGin/DNA and BCGsc/DNA vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, and DNA-HSP65. (B) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, DNA-HSP65 and BCGsc/DNA. ● BCGin vs BCGsc. (C) ◆ All immunized-infected mice vs Infected mice. * BCGin/DNA vs BCGsc, BCGin, DNA-HSP65. (D) ◆ All immunized-infected mice vs Infected mice. p < 0.05 was considered significant. Data are representative of two experiments.
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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 vaccin...
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... One of the main products under development by our group is a DNA vaccine containing the gene that expresses the Mycobacterium leprae hsp65 antigen (DNA-hsp65) with broad immunomodulatory and/or immunoregulatory properties [2]. Our previous studies have demonstrated that DNA-hsp65 confers a high level of protection in mice both prophylactically and therapeutically [2]: it works in prevention; can be used as a BCG booster [3]; can be used alone or in association with antimycobacterial drugs to completely control chronic TB, multidrug-resistant (MDR)-TB and latent TB (LTB) infection; and can prevent reactivation of infection [4][5][6][7]. ...
A Bacille Calmette–Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime–boost regimen, to help the induction of a stronger cellular immune response.
... One of the main products under development by our group is a DNA vaccine containing the gene that expresses the Mycobacterium leprae hsp65 antigen (DNA-hsp65) with broad immunomodulatory and/or immunoregulatory properties [2]. Our previous studies have demonstrated that DNA-hsp65 confers a high level of protection in mice both prophylactically and therapeutically [2]: it works in prevention [3]; can be used as a BCG booster [4]; acts in the immunotherapy of chronic TB, MDR-TB and LTB [5][6][7], it can be used concomitantly with antimycobacterial drugs to enhance therapeutic and immunotherapeutic action [7,8]; prevents reactivation of infection [5,6], and can act in those animals with compromised immune systems as in helminth coinfections [9][10][11]. ...
... Blood samples were collected via femoral venipuncture into Vacutainer tubes. 4 Animals were submitted to X-ray analysis 30-45 days before the first vaccination and after the last vaccination. ...
A Bacille Calmette-Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-days intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low, and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provided limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime-boost regimen, to help the induction of a stronger cellular immune response.
... DNA vaccines, with facile preservation, transport, manufacture, good stability, and efficient immune responses, are a useful strategy for the treatment and prevention of MDR-TB (Kutzler & Weiner, 2008). Currently, prophylactic and therapeutic DNA-vaccine vectors for delivery include hemagglutinating virus of Japan (HVJ)-liposomes and their envelopes, adenoviral or lentiviral vectors, and adeno-associated viral vectors (Okada et al. 2007, Yoshida et al., 2006; the DNA-vaccine antigens are HSP65 (Goncalves et al., 2007), MPT64 (Kamath et al., 1999), Ag85A (Tanghe et al., 2001), ESAT6 (Wang et al., 2004), Mtb10, and Mtb41 (Skeiky et al., 2000). DNA vaccines recombine the foreign gene encoding a specific antigen protein into a eukaryotic expression element to form recombinant plasmid DNA (Liang et al., 2008). ...
Vaccinations, especially DNA vaccines that promote host immunity, are the most effective interventions for tuberculosis (TB) control. However, the vaccine delivery system exhibits a significant impact on the protective effects of the vaccine. Recently, effective nanomaterial-based delivery systems (including nanoparticles, nanogold, nanoliposomes, virus-like particles, and virus carriers) have been developed for DNA vaccines to control TB. This review highlights the historical development of various nanomaterial-based delivery systems for TB DNA vaccines, along with the emerging technologies. Nanomaterial-based vaccine delivery systems could enhance the efficacy of TB vaccination; therefore, this summary could guide nanomaterial selection for optimal and safe vaccine delivery, facilitating the design and development of highly effective TB vaccines.
... The presence of immunodominant protein Ags, eliciting protective IRs in the acute phase of the infection highlights the interest in the CEC, as exemplified by the incorporation of this class of Ags in various vaccine platforms evaluated either in animal models or clinical evaluation [111,140,141,157,159,174,178,179,188,190,202,216,217]. ...
Even after decades searching for a new and more effective vaccine against tuberculosis, the scientific community is still pursuing this goal due to the complexity of its causative agent, Mycobacterium tuberculosis (Mtb). Mtb is a microorganism with a robust variety of survival mechanisms that allow it to remain in the host for years. The structure and nature of the Mtb envelope play a leading role in its resistance and survival. Mtb has a perfect machinery that allows it to modulate the immune response in its favor and to adapt to the host's environmental conditions in order to remain alive until the moment to reactivate its normal growing state. Mtb cell envelope protein, carbohydrate and lipid components have been the subject of interest for developing new vaccines because most of them are responsible for the pathogenicity and virulence of the bacteria. Many indirect evidences, mainly derived from the use of monoclonal antibodies, support the potential protective role of Mtb envelope components. Subunit and DNA vaccines, lipid extracts, liposomes and membrane vesicle formulations are some examples of technologies used, with encouraging results, to evaluate the potential of these antigens in the protective response against Mtb.
... [9][10][11][12] We have also described additional strategies to optimize the protective efficacy of this vaccine in pre-clinical assays such as prime-boost vaccination using BCG priming and DNA-HSP65 boosting, aggregates of DNA-HSP65 and cationic liposomes, or a single-shot vaccine formulation made up of DNA-HSP65, recombinant Hsp65 protein (rHsp65) and PLGA microspheres. [13][14][15][16] In an attempt to evaluate the immune stimulatory effects of DNA-HSP65 in human cells, we showed that monocytederived macrophages stimulated with DNA-HSP65 had increased production of TNF-a and were able to restrict bacterial growth. 17 A phase I clinical trial to establish the safety of DNA-HSP65 immunotherapy in patients with advanced head and neck squamous cell carcinoma has also been completed. ...
... 36 It is noteworthy that preclinical studies showed that protection induced by DNA-HSP65 immunization was dependent on upregulation of IFN-g levels, although this vaccine also had stimulated antigen-specific IL-10 production. 13,37 Our findings show that rHsp65 induced an increased secretion of IL-10 and DNA-Hsp65 stimulation resulted in an increase of IL-10-producing cells from healthy individuals and from TB patients. ...
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.
... The mycobacterial 65-kDa heat shock protein (HSP65), structurally and functionally similar to the eukaryotic HSP70 chaperon, has been identified as the major clinically important antigen of M. tb (50,51). Moreover, the HSP65 antigen is considered to be highly relevant to a subunit vaccine design against mycobacteria (52)(53)(54). It has been previously reported that fusion of epitopes to the C-terminal domain of HSP65 could successfully induce the antigen-specific humoral, cytotoxic T-lymphocyte (CTL) and Th1 response (55). ...
DNA-based vaccine is a promising candidate for immunization and induction of a T cell-focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi-functional T response against multi-TB antigens, a multi-epitope DNA vaccine and a “protein backbone grafting” designing method is adopted to graft 5 discontinuous T cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with 5 T cell epitopes derived from ESAT-6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was intramuscularly injected to C57BL/6 mice with 4 doses of 50µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope-scaffold construct was significantly more potent to induce IFN-γ+ T response to 5 T cell epitopes protein than other DNA constructs (with epitopes alone or with epitopes series connected to HSP65), especially in multi-functional-CD4+ T response. It also enhanced granzyme B+CTL and IL-2+CD8+ T response. In consistency, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi-epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.
... We then asked how Hsp65 would participate in the downmodulation of Th2 inflammation. Previous studies have shown that M. leprae Hsp65 may have an immunogenic or modulatory effect, depending on the microenvironment, formulation and adjuvant [16,[25][26][27]. ...
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.
Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy.
Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat shock protein (Hsp65) combined with CpG.
The treatment of mice with established allergy led to attenuation of eosinophilia, Th2 cytokines and airway hyperresponsiveness. Hsp65 plus CpG treatment also induced an increase in OVA-specific IFN-γ levels and in the frequency of lung inflammatory monocytes. Moreover, we show that the reduction of eosinophilia and the recruitment of inflammatory monocytes to the lungs required early triggering of TLR9, IFN-γ and CCR2 by immunotherapy components.
In addition to immune deviation to a Th1 response in the modulation of Th2 allergic inflammation, our findings also attribute an important role to the innate response mediated by TLR9, associated with the recruitment of CCR2-dependent monocytes.
Our findings show that Hsp65/CpG treatment is a promising strategy for consideration in translational studies. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Subsequently other studies have suggested the use of a wide range of immunogenic antigens from M. tuberculosis for use in DNA vaccines, such as HSP65 [30], MPT64 [31], Ag85A [32,33], ESAT6 [34], Mtb10 and Mtb41 [35] (Table I). Figure 2. The myocyte and/or keratinocyte that is transfected by the DNA vaccine expresses the antigen which is recognized by the dendritic cell. The dendritic cell processes the antigen and presents it to the CD8 ϩ cytotoxic T lymphocyte (CTL) cell through MHC-I and/or to CD4 ϩ through MHC-II. ...
... The results indicated that the DNA-HSP65 vaccine signifi cantly reduced CFUs, similarly to BCG, but not better. When the HSP65 DNA vaccine was used as a booster for BCG, although there was a decrease in bacterial burden in the lung, there were no signifi cant differences when compared to the mice vaccinated only with BCG [30]. Other studies showed that C57BL/6 mice immunized with 50 μ g of DNA vaccine encoding the 23 kDa protein (MPT64) and challenged with the H37Rv M. tuberculosis strain had a decreased bacterial burden in the lung when compared with non-vaccinated mice; however the BCG vaccinated mice showed better results in decreasing lung M. tuberculosis loads. ...
It is estimated that there are approximately eight million new cases of active tuberculosis (TB) worldwide annually. There is only 1 vaccine available for prevention: bacillus Calmette-Guérin (BCG). This has variable efficacy and is only protective for certain extrapulmonary TB cases in children, therefore new strategies for the creation of novel vaccines have emerged. One of the promising approaches is the DNA vaccine, used as a direct vaccination or as a prime-boost vaccine. This review describes the experimental data obtained during the design of DNA vaccines for TB.
... In this context, we observed that pVAXhsp65 and BCG similarly primed neonate mice for a strong immune response to pVAXhsp65 boosters administered later, at the adult stage [21]. Prime-boost strategies combining these two vaccines were also able to protect mice and guinea pigs against experimental TB [22] [23]. One of the arguments against the potential use of pVAXhsp65 alone or combined with BCG is the fact that hsp65 from M. leprae, whose gene is inserted in this DNA vaccine, presents a high degree of homology with its equivalent mammalian protein [24]. ...
A prime-boost strategy conserving BCG is considered the most promising vaccine to control tuberculosis. A boost with a DNA vaccine containing the mycobacterial gene of a heat shock protein (pVAXhsp65) after BCG priming protected mice against experimental tuberculosis. However, anti-hsp65 immunity could worsen an autoimmune disease due to molecular mimicry. In this investigation, we evaluated the effect of a previous BCG or BCG/pVAXhsp65 immunization on experimental autoimmune encephalomyelitis (EAE) development. Female Lewis rats were immunized with BCG or BCG followed by pVAXhsp65 boosters. The animals underwent EAE induction and were daily evaluated for weight loss and clinical score. They were euthanized during recovery phase to assess immune response and inflammatory infiltration at the central nervous system. Previous immunization did not aggravate or accelerate clinical score or weight loss. In addition, this procedure clearly decreased inflammation in the brain. BCG immunization modulated the host immune response by triggering a significant reduction in IL-10 and IFN- γ levels induced by myelin basic protein. These data indicated that vaccination protocols with BCG or BCG followed by boosters with pVAXhsp65 did not trigger a deleterious effect on EAE evolution.
... We previously reported that both DNAhsp65 and BCG could prime newborn mice for stronger immune responses to DNAhsp65 boosters at the adult stage [13]. Prime-boost strategies combining these two vaccines were also able to protect well-nourished mice and guinea pigs against TB infection [14,15]. More recently, we demonstrated that the antibody induction by the same genetic vaccine (DNAhsp65) was abrogated in animals submitted to dietary restriction [16]. ...
Cellular immunity is critical for protection against tuberculosis, but its integrity is compromised during undernutrition. The present study was designed to evaluate if the attenuated mycobacterium BCG is a safe vaccine for undernourished individuals. An experimental model of undernutrition was established by subjecting BALB/c mice to dietary restriction. These animals received 70% of the amount of food consumed by the healthy control group and exhibited physiological alterations compatible with malnutrition, including body weight loss, reduced levels of triglycerides and glucose, and reduced lymphocyte numbers. Undernourished mice were immunized with BCG, and the mycobacterial loads in lymph nodes, spleen, liver, lungs, and thymus were determined. A much higher proportion of undernourished mice exhibited bacterial dissemination to the lymph nodes, spleen and liver. In addition, only undernourished animals had bacteria in the lungs and thymus. Concomitant with higher mycobacterial loads and more widespread BCG dissemination in undernourished mice, production of TNF-α, IFN-γ, and IL-10 was also diminished in these mice. Taken together, these results indicate that BCG infection is more severe in undernourished mice. Whether a similar phenomenon exists in undernourished children or not remains to be thoroughly investigated.
