Vaccine 28 (2010) 1528–1534
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HSP65 DNA as therapeutic strategy to treat experimental paracoccidioidomycosis Alice M. Ribeiro a , Anamelia L. Bocca a,b , André C. Amaral a,c , Ana Camila C.O. Souza a , Lúcia H. Faccioli d , Arlete A.M. Coelho-Castelo d , Florêncio Figueiredo a,c , Célio L. Silva e,f , Maria Sueli S. Felipe b,∗ a
Laboratory of Pathology, Faculty of Medicine, University of Brasília, 70910-900 Brasilia, DF, Brazil Biological Sciences Institute, University of Brasília, 70910-900 Brasilia, DF, Brazil c Genomic Science and Biotechnology, Catholic University of Brasilia, 70790-160 Brasília, DF, Brazil d Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903 Ribeirão Preto, SP, Brazil e Farmacore Biotecnology Ltd., 14049-900 Ribeirão Preto, SP, Brazil f Center for Tuberculosis Research, School of Medicine of Ribeirão Preto, Millennium Institute of Network-TB, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 28 August 2009 Received in revised form 13 November 2009 Accepted 20 November 2009 Available online 31 December 2009 Keywords: Immunotherapy DNAhsp65 Paracoccidioidomycosis
a b s t r a c t The conventional treatment for paracoccidioidomycosis, the most prevalent mycosis in Latin America, involves long periods of therapy resulting in sequels and high frequency of relapses. The search for new alternatives of treatment is necessary. Previously, we have demonstrated that the hsp65 gene from Mycobacterium leprae shows prophylactic effects against murine paracoccidioidomycosis. Here, we tested the DNAhsp65 immunotherapy in BALB/c mice infected with Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. We observed an increase of Th1 cytokines accompanied by a reduction in fungal burden and pulmonary injury. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Paracoccidioidomycosis (PCM) is a health problem in Latin America where an estimated 10 million individuals may be infected by its etiological agent, the dimorphic human pathogenic fungus Paracoccidioides brasiliensis [1]. PCM is the most prevalent systemic endemic mycosis in South America notably in Brazil, Colombia, Venezuela, and Argentina [2]. The fatal acute PCM affects the reticule endothelial system, whereas the chronic PCM affects mainly the lung, which shows a granulomatous inflammation with an inefficient cellular immune response [1,3]. The conventional therapy for PCM is based on sulphanamides, amphotericin B and azole derivatives [4]. Long-term therapy is usually required to warrant a good clinical response and avoid relapses. However, the treatment has its limitations – for example, high toxicity, low efficiency and drug resistance, mainly because of the growing number of immunocompromised patients. Thus, despite the existing antifungal drugs for treating patients with PCM, there is a demand for new therapeutical interventions that could help the clinicians to approach severely ill patients with an impaired
∗ Corresponding author at: Dept. of Cell Biology – Institute of Biology, University of Brasília, 70910-900 Brasilia, DF, Brazil. Tel.: +55 61 33072423; fax: +55 61 33498411. E-mail address:
[email protected] (M.S.S. Felipe). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.11.062
host cell response, who usually have a poor and/or late response to conventional antifungal therapy. Because of the escalating number of immunocompromised patients, such as those infected with HIV or undergoing anticancer therapy, the cases of resistance are increasing, which calls for even higher doses of the antifungal agents in use today. Higher dosages of some antifungal agents may cause serious toxic side effects, such as nephrotoxicity [5]. For that reason, alternative therapies are being studied using candidate antigen molecules and its mechanisms of protection against various fungi, including P. brasiliensis [6]. Among these molecules, heat-shock proteins (HSPs) represent attractive candidates due to their association with both innate and adaptive immunity [7]. HSP molecules have been applied into DNA- or protein (peptide)-based vaccines as antigens, chaperones or adjuvants [8]. Numerous members of this family of proteins have been tested for prophylaxis or immunotherapy against a great variety of illnesses, for instance, tumors, autoimmune diseases, and several types of infections, including mycosis [9–11]. In both human and experimental fungal infection, cell-mediated immunity is critical for host defense [12]. The successful resolution of infection with P. brasiliensis is dependent on the activation of cellular immunity. The immune response towards a preferential Th1 activation, with IFN-␥ production and efficient macrophage activation is able to contain fungal dissemination and disease progression [13].
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In animal models, DNA vaccines have been successfully used against various pathogens, such as protozoa (Trypanosoma cruzi [14], Leishmania major [15] and Giardia lamblia [16], viruses (hepatitis B) [17], mycobacteria (Mycobacterium tuberculosis) [18]. Current studies indicate that the HSPs induce preferentially a cellular immune response [19] and HSP vaccines can be used for cancer immunotherapy [20] as well as for the treatment of tuberculosis [21]. Several studies have demonstrated a potential use of HSP65 from Mycobacterium leprae as an immunomodulator. A DNA vaccine encoding for the protein HSP65 (DNAhsp65) was able to confer protection to mice and guinea pigs against M. tuberculosis challenge [22]. Other experiments showed even better results when DNAhsp65 and drug treatments were combined [21]. In both cases, therapeutic effects appeared to be associated with the priming of a strong Th1 immune response and down-regulation of Th2 cytokines. More importantly, this response not only reduced bacilli loads in the lungs but also conserved the histological structure of the lung parenchyma [22]. Our previous results have shown that a DNAhsp65 vaccine protected mice against a virulent strain of P. brasiliensis, the etiological agent of the PCM [6]. In the present study, we used the DNAhsp65 plasmid to treat mice infected with the virulent P. brasiliensis. The immunomodulation and the ability to elicit specific cellular and humoral immunity by DNAhsp65 as well as its use as immunotherapeutic were investigated. 2. Methods 2.1. DNAhsp65 plasmid construction and purification The plasmid DNAhsp65 was derived from the vector pVAX1 vector (Invitrogen, Carlsbad, CA, USA), which had previously been digested with BamHI and NotI (Invitrogen), and a fragment of the M. leprae hsp65 gene was inserted. The empty pVAX vector was used as a control. Plasmid DNA was obtained from transformed DH5␣ E. coli cultured in LB liquid medium (Gibco BRL, Gaithersburg, MD, USA) containing kanamycin (50 g/mL). The plasmids were purified using the Endofree Plasmid Mega kit (Qiagen, Valencia, CA, USA). DNA concentration was estimated spectrophotometrically at 260 and 280 nm using the Gene Quant II apparatus (Pharmacia Biotech, Buckinghamshire, UK). Endotoxin levels were determined using a QCL-1000 Limulus amoebocyte lysate kit (Cambrex Company, Walkersville, MD, USA) and were less than 0.1 endotoxin units (EU)/g, as recommended by European and US Pharmacopoeias. The purity of DNA preparations was verified by electrophoresis (1% agarose). 2.2. Mice Male BALB/c mice (6–8-week old) were obtained from University of São Paulo (Ribeirão Preto Campus, SP, Brazil) and maintained under standard laboratory conditions. All experiments involving animals were approved by Bioethical Committee of University of Brasília (UnB, Brasília, DF, Brazil) and conducted in accordance with their guidelines. 2.3. P. brasiliensis strain and infection The yeast form of the virulent P. brasiliensis strain 18 [23] was used for the infection assays. The fungus was cultured in liquid YPD medium (w/v: 2% peptone, 1% yeast extract, 2% glucose) at 36 ◦ C in a rotary shaker (220 rpm) for 5 days. After this period, a suspension of P. brasiliensis cells was prepared at a concentration of 107 viable cells/mL. Viability was determined through the Janus Green B vital
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dye method [24] (Merck, Darmstadt, Germany). It was found to be higher than 80%. To infect the mice intravenously, 100 L of this suspension containing 106 cells was inoculated per animal. The chosen route of infection was intravenous because this is the established model of stimulating systemic and chronic diseases and that is the situation we intended to analyze with respect to the effectiveness of the DNAhsp65 plasmid therapy. 2.4. Treatment After 30 days of infection, the treatment was initiated by intramuscular route. Mice were divided into four groups (10 animals per group): group I – mice non-infected and non-treated (noninfected group); group II – mice infected and treated with saline 0.9% (infected group); group III – mice infected and treated with 4 doses of 100 g of empty vector pVAX1 at 2-week intervals (pVAX1 group); group IV – mice infected and treated with 4 doses of pVAX1hsp65 at 2-week intervals (DNAhsp65 group). The animals were euthanized by cervical dislocation 15 days after the last treatment dose. Lung, spleen, liver and serum samples were collected for sequential analysis. 2.5. Histopathology and determination of P. brasiliensis CFU in lungs To evaluate lesion progress, lung tissue fragments were fixed in 10% formalin for 6 h, dehydrated in alcohol, and embedded in paraffin. Serial 5-m sections were stained either with hematoxylin–eosin (HE) to visualize the fungus and granulomatous appearances or with Masson’s trichrome to detect collagen fibers. Lung fungal burdens were measured by quantitative counts of colony-forming units (CFU) of P. brasiliensis. Lung, spleen and liver fragments were homogenized in 1.0 mL of PBS (pH 7.2). Aliquots of 100 L of these homogenates were plated onto brain heart infusion agar (BHI agar), supplemented with 4% horse serum, 5% P. brasiliensis 192 (Pb192) culture filtrate [25] and gentamycin 40 mg/L (Gentamycin Sulfate, Schering-Plough, Rio de Janeiro, Brazil). The Pb192 culture filtrate was prepared as previously described [25]. The plates were incubated at 36 ◦ C, and the number of colonies was counted after incubation for 21 days. Results were expressed as number of CFU ± standard error of the mean (S.E.M.) per gram of lung tissue. 2.6. Cell proliferation assay and measurement of cytokines concentration T-cell proliferative responses to concanavalin-A (ConA) were studied by [3 H]-thymidine incorporation as previously described [26]. Spleen cells were disrupted in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, 5% non-essential amino acids (Sigma Chemical Co., St. Louis, MO), 2 M streptomycin (100 g/mL), and 5% fetal bovine serum (FBS). Cells were washed twice in serum-free RPMI, counted, added to 96-well plates at a cell density of 3 × 105 cells/well and subsequently stimulated with ConA (4 g/mL, Sigma Chemical Co., St. Louis, MO). The experiments were performed in triplicate at a final volume of 200 L/well. After 48 h of incubation at 37 ◦ C under 5% CO2 , cultures were pulsed for 12 h with 1 Cie/well of with H3 -labeled thymidine (Amersham, Arlington Heights, IL) and then harvested. The incorporation of H3 -thymidine was measured using a Liquid Scintillation Counter (Beckman Instruments); the data were expressed as means ± S.E.M. of counts per minute of H3 -thymidine incorporation. Supernatants of spleen cells cultured from experimental groups were used for detection of cytokines production. The cytokines
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interleukin-4 (IL-4), -10 (IL-10), -12 (IL-12), and interferon-gamma (IFN-␥) were measured using a commercial ELISA kit (according to the guidelines established by BD Biosciences, San Diego, CA). The cytokine levels present in the supernatant preparations obtained from spleen cell culture were calculated based on a standard curve provided by the commercial kit. Data were expressed as the mean(log10 ) ± S.E.M. 2.7. Anti-HSP65 specific antibodies detection The specific IgG1 and IgG2a isotypes were measured by Enzyme Linked ImmunoSorbent Assay (ELISA). Briefly, 96-well plates were sensitized with rHSP65 protein (250 ng/100 L well) overnight at 4 ◦ C. Plates were blocked with 1% bovine serum albumin in PBS for 60 min at 37 ◦ C and then, serum samples (1:100 dilution) were applied for 2 h at 37 ◦ C. After washing with PBS 0.05% Tween 20, peroxidase-labeled antibodies specific for mouse IgG1 or IgG2a isotypes (Sigma Chemical Co., St. Louis, MO) were diluted to 1:5000 and incubated for 2 h at 37 ◦ C. The plates were washed seven times with PBS 0.05% Tween 20 and incubated with H2 O2 and Ophenylenediamine for the reaction. After the addition of 20 L of H2 SO4 , (2N) (stop solution) the reactions were read at 490 nm in an ELISA reader (BioRad, model 2550, CA, USA). 2.8. Measurement of nitric oxide (NO) concentration The concentration of nitrite (NO2 − ) in spleen cell supernatants was measured by Griess assay as described previously [27]. NO2 concentration in culture supernatants was used as an indicator of NO generation and measured with the Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, 2.5% H3 PO4 ). Briefly, 100 l of the culture supernatants was added to an equal volume (v/v) of Griess reagent and was incubated at room temperature for 10 min. Absorbances were measured at 540 nm using a microplate reader. The NO2 − concentration was determined using a standard curve of 1–200 M of NaNO2 . The conversion of absorbance to M of NO was deduced from a standard curve by using a known concentration of NaNO2 diluted with RPMI medium. All determinations were performed in triplicate and expressed as the mean ± S.E.M. of M of NO2 . The serum nitrate concentration was determined by reducing nitrate to nitrite enzymatically using nitrate reductase as described previously [28]. The total amount of nitrite was then determined using the Griess method. The results are reported as M of NO3 . 2.9. Experimental reproducibility and statistical analysis All experiments were repeated three times independently. Differences between experimental groups were analyzed by one-way ANOVA test and multiple comparisons with Dunnet’s post-test. All calculations and plotting were done using PRISM 5.0 (Graph Pad Software for Science, San Diego, CA, USA). P values were considered significant at P < 0.05. All values are means ± S.E.M. 3. Results 3.1. Pulmonary histopathology and fungal burden in lungs, liver and spleen from animals infected with P. brasiliensis and treated with DNAhsp65 Pulmonary histological examination of animals from infected and pVAX1 groups revealed a diffuse and intense granulomatous inflammatory infiltrate containing mononuclear cells, epithelioid cells, multinucleated giant cells and numerous fungal yeast cells (Fig. 1A). Progression of the infection was observed mainly in the lungs and was characterized by a diffuse inflammatory response
possibly in response to the presence of P. brasiliensis in this organ from both groups, as demonstrated in the fungal burden. In contrast, the histological examination of lungs from the DNAhsp65 group showed focal and discrete granulomatous inflammatory infiltrate containing mononuclear cells, small granulomas and sparce fungal yeast cells (Fig. 1B). The deposition of collagen (fibrosis) was also more intense in the lung tissue of infected and pVAX1-treated groups (Fig. 1C) when compared with DNAhsp65 group (Fig. 1D). The decrease in the number of yeast cells by the DNAhsp65 treated group was confirmed by P. brasiliensis CFU quantification in lung, liver and spleen. The fungal load recovered from DNAhsp65 group was nearly half of that observed in the infected and pVAX1 groups (Fig. 1E). The pVAX1 group showed a fungal burden similar to that of the infected group. 3.2. Effect of DNAhsp65 treatment on lymphoproliferation and determination of the cytokine profile In order to determine if the immune response of the treated animals was modulated by DNAhsp65, we examined splenocytes proliferation and cytokine production. High level of splenocytes stimulation was induced by the DNAhsp65 treated mice in the presence of ConA. Similar results were found for the non-infected group (Fig. 2A). Fig. 2A also shows that mice from the infected and pVAX1 groups displayed comparatively low ability to proliferate in the presence of stimuli. Cytokine production was examined by monitoring the levels of IL-4, IL-10, IL-12 and IFN-␥ in culture supernatants of splenocytes from the experimental groups. Mice treated with DNAhsp65 produced significantly higher levels of IFN-␥ and IL-12 and similar levels of IL-4 and IL-10 compared to infected mice (Fig. 2B). The production of cytokine in the infected and pVAX1 groups was similar to that of the non-infected group. 3.3. Humoral immune response and NO production of mice infected with P. brasiliensis and treated with DNAhsp65 To characterize the mechanisms responsible for the protective effects of DNAhsp65, we investigated the antibody response to HSP65 in BALB/c mice infected with the virulent P. brasiliensis, after receiving the different treatments. Anti-HSP65 antibodies were detected in the sera of mice from the infected and pVAX1 groups at significantly lower levels than in mice treated with DNAhsp65, although there were no differences between IgG1 and IgG2 (Fig. 3A). The treatment with DNAhsp65 increased the level of antibodies anti-hsp65, which indicates that the protein was able to stimulate the immune system. We evaluated the production of NO by measuring the concentrations of NO2 and NO3 in the spleen cell cultures supernatants and serum, respectively. The animals treated with DNAhsp65 showed much higher amounts of both NO2 and NO3 than the animals belonging to the non-infected group. The controls groups, infected and pVAX1, did not present alterations in the amounts of NO2 and NO3 when compared to the non-infected group (Fig. 3B). Together, these data suggest that the DNAhsp65 induces the immune response of mice and is able to not only protect the animals against the infection with P. brasiliensis, but also treat them for PCM. 4. Discussion Considering the increase in the worldwide incidence of fungal infections, the development of new therapies for these diseases has become of great importance to public health [29]. The current
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Fig. 1. Histopathological images showing pulmonary fungal burden in animals from the pVAX1 and DNAhsp65 groups after 30 days of infection with P. brasiliensis. (A) Intense granulomatous inflammatory infiltrate containing mononuclear cells, epithelioid cells, multinucleated giant cells and numerous fungal yeast cells in animal of the pVAX1 group (HE, 200×). (B) Discrete granulomatous inflammatory infiltrate containing mononuclear cells, small granuloma and sparse fungal yeast cells in animal of the DNAhsp65 group (HE, 200×). (C) Deposition of collagen (fibrosis) in the lung tissue of an animal from the pVAX1 group (Masson’s trichrome, 200×). (D) Masson’s staining for collagen – animal of the DNAhsp65 group (200×). Fungal burden recovery from lungs, liver and spleen of mice infected with 106 cells of P. brasiliensis (E). The animals were treated with DNAhsp65 after 30 days of infection. The bars depict means ± S.E.M. of CFU obtained from experimental groups (infected, pVAX1 and DNAhsp65) after 30 days of infection with P. brasiliensis. *P < 0.05 in relation to the infected group.
therapy to treat mycosis is based on polienes and azoles, depending on the severity of the infection [4]. To treat severe cases of PCM amphotericin B followed by itraconazole and sulfametoxazole are indicated. The main disadvantage of using amphotericin B is its occasional toxicity. In the case of itraconazole or sulfametoxazole, the long period of treatment required may cause patients to quit medication, possibly leading to recurrence of the disease [4]. Given these difficulties, new approaches to the prevention and the treatment of systemic fungal infections need to be developed. Alternative strategies to conventional chemotherapy for fungal diseases have been explored. Ajoene, a compound derived from garlic, demonstrated, in vitro, antifungal activity against Aspergillus
niger and C. albicans [30]. It also inhibited the in vitro growth of P. brasiliensis, in vitro, by causing changes in the plasma membrane structure but not the cell wall [31]. Recently, Thomaz et al. showed that ajoene therapy was effective in Balb/c mice infected with P. brasiliensis [32]. Similarly, patients of PCM who received glucan had a stronger and more favorable response to therapy, although they were seriously ill [33]. Combined therapy of immunotherapeutics and antifungal agents in the treatment of PCM has been investigated. The peptide P10, a 15-amino acid peptide identified in the glycoprotein Gp43, the major diagnostic antigen of PCM, elicits the secretion of IFN-␥ and other Th1 type cytokines, resulting in the protec-
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Fig. 2. Levels of IL-4, IL-10, IL-12 and IFN-gamma produced by spleen cells (A) and levels of IgG2a and IgG1 isotype levels (B) in sera of mice infected with 106 cells of P. brasiliensis and then treated with DNAhsp65. (A) The bars depict means ± S.E.M. of cytokine production by spleen cells from mice of the experimental groups (noninfected, infected, pVAX1 and DNAhsp65). (B) The bars depict levels of anti-rHsp65, IgG2a and IgG1 isotypes ± S.E.M. from experimental groups (non-infected, infected, pVAX1 and DNAhsp65) after 30 days of infection. *P < 0.05 in relation to infected group.
tion against this mycosis [34]. Its administration, as an adjuvant to the chemicals used in the PCM therapy, showed that P10 improves the therapeutic effectiveness of some antifungal agents [34]. This combined treatment showed to be an important procedure to improve treatment of PCM and, most importantly, to prevent relapse. Microparticles will probably play an important role in future chemotherapy too. Recently, our group has tested amphotericin B in PLGA nanoparticles and it was effective against P. brasiliensis [35]. HSPs are recognized as important molecules in the modulation of the immune system being able to activate a cellular immune response [19]. In experimental models, HSP-based vaccines have been tested for various diseases [10,15,17,21]. Clinical trials are being conducted. Recently, Roman et al. showed that the vaccine with the HSP-7 induced lesions regression in patients with high intraepithelial neoplasia [36]. In the case of fungal diseases, different genes have been investigated as to their ability to elicit a protective immune response against H. capsulatum, P. brasiliensis, Blastomyces dermatitidis, Cryptococcus neoformans and Coccidioides immintis [37]. In an experimental model of candidiasis, antibody protection can occur through the recognition of specific proteins, such as HSP90 [38]. The recombinant HSP60 (rHSP60) induces resistance in immunocompetent animals infected with H. capsulatum. CD4+ T cells and a Th1 cytokine profile are needed for this resistance [39]. In both human and experimental fungal infection, cell-mediated immunity is critical for host defense [12,37,40]. Cellular immune response plays an important role in the resistance to P. brasiliensis infection [14]. Immunological studies on patients with polarized
Fig. 3. Lymphoproliferation of cells in response to stimulation with concanavalinA (ConA) (A) and NO generation in spleen cells culture (NO2 ) and serum (NO3 ) from mice infected with 106 P. brasiliensis and then treated with DNAhsp65 (B). (A) Spleen cells were isolated from experimental groups (non-infected, infected, pVAX1 and DNAhsp65) after 30 days of infection and cultured in presence of RPMI medium or ConA (4 g/mL). The bars depict means ± S.E.M. of counts per minute of H3 -thymidine incorporation by spleen cells of the experimental groups. (B) The bars depict means ± S.E.M. of NO production by cell culture (NO2 ) and serum (NO3 ) from experimental groups (non-infected, infected, pVAX1 and DNAhsp65) after 30 days of infection. *P < 0.05 in relation to infected group.
forms of PCM show an association between Th1 based reactivity and asymptomatic or mild forms of infection. In contrast, Th2 cell response correlates with severe disease. Patients with systemic PCM tend to show depressed cellular immune responses, compared to those with localized disease [40]. Several clinical observations indicate an inverse relationship between IFN-␥ and IL-10 productions in patients with fungal infections. High levels of IL-10, negatively affecting IFN-␥ production, are detected in chronic candidal disease [41], in the severe form of PCM [42] and in neutropaenic patients with aspergillosis [43]. Here we reported the efficacy of Mycobacterium HSP65 DNA in the treatment of a fungal model, the PCM. Infected animals treated with this vaccine experienced a reduction in the pulmonary fungal burden. Moreover, there was less lung tissue compromised by collagen deposition. This is an important finding because once the excessive deposition of collagen by fibroblasts is related to pulmonary fibrosis, one of the most serious consequences of PCM [44]. Animals infected with P. brasiliensis and subsequently treated with DNAhsp65 presented high levels of NO, which may have contributed to the elimination of the fungus since NO is an antimicrobial molecule of great importance in the defense against fungal pathogens, such as P.brasiliensis [26]. Moreover, the DNAhsp65 restored the lymphoproliferation ability of the animals, which was similar to that control animals (non-infected group). Spleen cell from mice infected with the virulent isolate P. brasiliensis 18
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have hampered their proliferation after stimulation with different mitogens [6,45]. On the pattern of cytokines, the treatment with DNAhsp65 induced an increase of Th1 cytokines levels, standard of protective response to PCM. No differences in the cytokine production pattern were observed between infected and non-infected animals, which is probably due to the absence of stimulation with specific mitogens at this period of infection and to the inherent genetic background of the chosen mouse strain. In the murine model, resistant mice are assumed to direct their immune response towards a preferential Th1 activation with production of IFN-␥, and efficient macrophage activation is able to contain fungal dissemination and disease progression [14]. In this work, the used formulation of DNAHSP65 for the treatment of PCM did not include adjuvants, which means that the immune response was probably specific against the corresponding protein. Recombinant HSP65 has been used for prime-boosting and enhancing the effect of the DNA vaccine itself [46]. These data support our previous results using the DNAhsp65 as vaccine to protect mice against P. brasiliensis [6]. The results presented here showed that the DNAhsp65 vaccine could be explored as an adjuvant for PCM therapy. The DNAhsp65 vaccine can lead to significant improvements – for instance, it may be able to stimulate the immune system for a long time without integrating into the genome of the host [15]. Based on our results, the association of DNAhsp65 with another antifungal agent should be utilized towards a new therapy against this and other mycoses. Acknowledgements We are grateful to Izaíra T. Brandão, Ana Masson and Viviane M. Leal for their technical assistance. The authors thank Dr. Fabiana P. Carneiro for her support in histopathological analyses. This work was supported by research grants from Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References [1] Brummer E, Castaneda E, Restrepo A. Paracoccidioidomycosis: an update. Clin Microbiol Rev 1993;6:89–117. [2] Restrepo A. The ecology of Paracoccidioides brasiliensis: a puzzle still unsolved. J Med Vet Mycol 1985;23:323–34. [3] de Camargo ZP, de Franco MF. Current knowledge on pathogens and immunodiagnosis of paracoccidioidomycosis. Rev Iberoam Mycol 2000;17:41–8. [4] Shikanai-Yasuda MA, Telles FQ, Mendes RP, Colonbo AL, Moretti ML. Guidelines in paracoccidioidomycosis. Rev Soc Bras Med Trop 2006;39(3):297–310. [5] Lemke A, Kiderlen AF, Kayser O. Amphotericin B. Appl Microbiol Biotechnol 2005;68:151–62. [6] Ribeiro AM, Bocca AL, Amaral AC, Faccioli LH, Galetti FCS, Zárate-Bladés CR, et al. DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection. Vaccine 2009;27(4):606–13, 22. [7] Segal BH, Wang XY, Dennis CG, Youn R, Repasky EA, Manjili MH, Subjeck JR. Heat shock proteins as vaccine adjuvants in infections and cancer. Drug Discov Today 2006;11:534–40. [8] Bolhassani A, Rafati S. Heat-shock proteins as powerful weapons in vaccine development. Expert Rev Vaccines 2008;7(8):1185–99. [9] Oglesbee MJ, Pratt M, Carsillo T. Role for heat shock proteins in the immune response to measles virus infection. Viral Immunol 2002;15:399–416. [10] Scheckelhoff M, Deepe Jr GS. The protective immune response to heat shock protein 60 of Histoplasma capsulatum is mediated by a subset of V8.1/8.2+ T cells. J Immunol 2002;169:5818–26. [11] Ferraz JC, Stavropoulos E, Yang M, Coade S, Espitia C, Lowrie DB, et al. A heterologous DNA priming-Mycobacterium bovis BCG boosting immunization strategy using mycobacterial Hsp70, Hsp65, and Apa antigens improves protection against tuberculosis in mice. Infect Immun 2004;72:6945–50. [12] Romani L. Immunity of fungal infections. Nat Immunol 2004;4(1):1–23. [13] Kashino SS, Fazioli RA, Cafalli-Favati C, Meloni-Bruneri LH, Vaz CA, Burger E, et al. Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-gamma production. J Interferon Cytokine Res 2000;20(1):89–97, 68:151162. [14] García GA, Arnaiz MR, Laucella SA, Esteva MI, Ainciart N, Riarte A, et al. Immunological and pathological responses in BALB/c mice induced by
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