Vaccine 27 (2009) 606–613

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DNAhsp65 vaccination induces protection in mice against Paracoccidioidesbrasiliensis infection

Alice M. Ribeiroa, Anamelia L. Boccaa,b, André C. Amaral c, Lucia H. Facciolid, Fabio C.S. Galetti e,Carlos R. Zárate-Bladés f, Florencio Figueiredoa,c, Célio L. Silvae,f, Maria Sueli S. Felipeb,∗

a Laboratory of Pathology, Faculty of Medicine, University of Brasília, Brasilia, DF 70910-900, Brazilb Biological Sciences Institute, University of Brasília, Brasilia, DF 70910-900, Brazilc Genomic Science and Biotechnology, Catholic University of Brasilia, Brasília, DF 70790-160, Brazild Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Rebeirão Preto, SP 14040-903, Brazile Farmacore Biotechnologia Ltda, Rebeirão Preto, SP 14049-900, Brazilf Center for Tuberculosis Research, School of Medicine of Rebeirão Preto, Millennium Institute of Network-TB, University of São Paulo, Rebeirão Preto, SP 14049-900, Brazil

a r t i c l e i n f o

Article history:Received 21 July 2008Received in revised form26 September 2008Accepted 2 October 2008Available online 24 November 2008

a b s t r a c t

Heat-shock proteins are molecules with extensive data showing their potential as immunomodulators ofdifferent types of diseases. The gene of HSP65 from Mycobacterium leprae has shown prophylactic andimmunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leish-maniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimentalmodel of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscularimmunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduc-

Keywords:DNA vaccineHP

tion of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 maybe an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment.

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. Introduction

Systemic mycosis are infections that are usually difficult to treat,or different reasons, including the generally chronic state of theisease at the moment of diagnosis, the great resistance of theathogens to many of the available drugs, and the long period ofherapy that represents high costs in terms of anti-fungal agentsnd disability because the patient cannot work [1].

This scenario is complicated even more by the higher incidencef these infections in developing world. Because the diagnosis isrequently made during the advanced stages of the disease whenhronic lesions are present, which occurs with Paracoccidioidesrasiliensis (P. brasiliensis) infection, a chronic granulomatous dis-ase has become endemic in Latin America with a high incidence in

razil, Colombia, Venezuela, and Argentina [2]. The data collectedy various researchers clearly describe a scenario where there isn urgent need to improve the therapeutic results of drugs andhe development of effective vaccines. For this reason, agents that

∗ Corresponding author at: Universidade de Brasilia, Department of Cell Biology,nstitute of Biology, 7000000 Brasilia, DF, Brazil. Tel.: +55 61 33072423;ax: +55 61 33498411.

E-mail address: msueli@unb.br (M.S.S. Felipe).

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.10.022

© 2008 Elsevier Ltd. All rights reserved.

an modulate the immune system are especially attractive candi-ates [1]. Among these molecules, heat-shock proteins (HSPs) arene of the most attractive options because they were associatedith different phenomena over innate and adaptive immunity [3,4].umerous members of this family of proteins have been tested forrophylaxis and/or immunotherapy against a great variety of ill-esses, such as tumors, autoimmune diseases, and several typesf infections, including mycosis [5–9]. The rationale underlyinghe use of HSPs in such different kinds of processes is the vari-ty of molecular phenomena in which they participate. Some ofhem are of the utmost importance for the following reasons: theirapacity to form a complex with pathogens and tumoral derivedeptides; their ability to be recognized by immune receptors likeoll-like (TLRs) and chemokine receptors; their involvement in theross-priming of antigens for cytotoxic T cells; and some pathogen-erived HSPs are immunodominant antigens [4,10].

Several articles have demonstrated the potential use of HSP65rom Mycobacterium leprae (M. leprae) as an immunomodula-or. This molecule was tested in the form of a genetic vaccine

DNAhsp65) and was able to confer protection to mice and guineaigs against Mycobacterium Tuberculosis (M. tuberculosis) challenge11,12]. Subsequent experiments showed that the therapy for activeuberculosis has better results when the association of DNAhsp65nd drugs is employed. In both cases, the benefit seems to be

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ssociated with the priming of a strong Th1 immune response andeduction of Th2 cytokines [13,14]. More importantly, the effectf this response not only reduces bacilli loads in lungs, but alsoonserves the histological structure of lung parenchyma [15]. Inddition, DNAhsp65 vaccination prevented infection of Leishma-ia major (L. major) [16]. Moreover, Santos-Junior et al. [17,18]howed that following DNAhsp65 vaccination, mice susceptible toeveloping arthritis or diabetes did not present with any signs ofomplication, rather a reduction of disease-associated lesions wasbserved in both cases. Finally, while the potential of HSP65 toown-modulate the proliferation of tumor cells was demonstrated5 years ago [19], more recently a Phase I clinical trial with headnd neck carcinoma patients showed that immunotherapy withNAhsp65 did not induce any relevant toxic effect. In this study

t was possible to observe a reduction in tumor mass in 4 of 21atients included in the trial [20].

Keeping this in mind, along with the potential of HSP65 asn immunodulating factor and the molecular precision of DNAmmunization in priming cellular and humoral immunity that isnrivaled by any other type of vaccine [21], we administered theNAhsp65 vaccine and performed subsequent challenges with P.rasiliensis. We observed a reduction in fungal burden, modulationf the immune response towards a Th1 profile, and the reductionf immunopathology in the lungs. These results provide a newrospective on the development of DNA-immunotherapy of Para-occidioidomycosis (PCM).

. Materials and methods

.1. DNAhsp65 vaccine

The plasmid pVAX1 (Invitrogen) containing the cytomega-ovirus (CMV) promoter and a cloned cDNA encoding the hsp65ene from M. leprae (pVAX1-hsp65 = DNAhsp65 vaccine) has beenreviously described [22]. The empty vector (pVAX1 withouthe hsp65 gene) was used as a control. Escherichia coli DH5�transformed with pVAX1 or with pVAX1-hsp65) was grown inuria Broth (LB, GIBCO-BRL) containing kanamycin (100 �g/mL).ndotoxin-free plasmid DNA was isolated using the endofree plas-id purification kit (QIAGEN AG, Basel, Switzerland). DNA plasmid

oncentrations were determined at 260 nm and 280 nm using aene quant II apparatus (Pharmacia Biotech, Buckingham-Shire,K). Endotoxin levels were determined using a QCL-1000 Limulusmoebocyte lysate kit (Cambrex Company, Walkersville, MD, USA)nd were less than 0.1 endotoxin units (EU)/�g, as recommendedy European and US Pharmacopoeias.

.2. Fungal strain and inoculum

P. brasiliensis strain 18 (Pb18) is highly virulent [23] and wasbtained from the fungal collection of the Laboratory of Moleculariology at the Biology Institute of University of Brasilia. The fungusas cultured in liquid YPD medium (w/v: 2% peptone, 1% yeast

xtract, 2% glucose) at 36 ◦C in a rotary shaker (220 rpm). After 5ays of growth, a suspension of P. brasiliensis cells was prepared atconcentration of 1 × 107 viable cells/mL. Viability was determinedith Janus Green B vital dye [24] (Merck, Darmstadt, Germany) andas always higher than 80%.

.3. Experimental design

Unless otherwise stated, BALB/c mice (male, 6–8 weeks old)ere obtained from University of São Paulo (Ribeirão Preto Campus,

P, Brazil) and maintained under standard laboratory conditions.ll immunizations were performed intramuscularly. Mice were

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27 (2009) 606–613 607

ivided into four groups (20 animals per group): (1) mice immu-ized with 3 doses of 100 �g of empty vector pVAX1 at 2-week

ntervals (pVAX1 group); (2) mice immunized with 3 doses ofVAX1-hsp65 at 2-week intervals (DNAhsp65 group); (3) mice

mmunized with saline (saline group); (4) non-immunized andon-infected mice (control group). Two weeks after the last vac-ine dose, mice were infected intravenously with 100 �L of theungal suspension described above. The chosen route of infectionas intravenous because this is the established model of simulating

ystemic disease, and that is the situation we intended to analyzeith respect to the effectiveness of the vaccine. Of the 20 animals

n each group, 10 were sacrificed after 30 days of fungal infectionnd the other 10 animals from each group were sacrificed after 60ays of infection. The animals were sacrificed by cervical disloca-ion. Lung, spleen and serum samples were collected for sequentialnalysis. All experiments were approved and conducted in accor-ance with guidelines of the Bioethical Committee of University ofrasilia (UnB), Brasília, DF, Brazil.

.4. Histopathological analysis and fungal burden determinationn lungs

For evaluation of lesion progress, animals from different groupsere sacrificed at the scheduled times. Lung fragments were

emoved and fixed in 10% formalin for 6 h, dehydrated in alcohol,nd embedded in paraffin. Serial 5-�m sections were stained withematoxylin–eosin (HE) to visualize the fungus and granulomatousppearances with masson trichrome to quantify collagen and silveror fungal evaluation.

The fungal burden in the lungs was measured by the num-er of CFU (colony-forming units) of P. brasiliensis. Lung fragmentsere homogenized in 1.0 mL of cold PBS (pH 7.2). A total of 100 �L

f the homogenates were plated onto brain heart infusion agarBHI agar), supplemented with 4% horse serum, 5% P. brasiliensis92 (Pb192) culture filtrate, and gentamicyn 40 mg/L (Gentamycinulfate, Schering-Plough, Rio de Janeiro, Brazil). The Pb192 cul-ure filtrate was prepared according to a published method [25].he plates were incubated at 36 ◦C, and the number of coloniesas counted after incubation for 7–21 days in plastic bags torevent desiccation [26]. Results were expressed as number ofFU ± standard error of the mean (S.E.M.) per gram of lung tissue.

.5. Analysis of anti-HSP65-specific antibodies

The specific IgG1 and IgG2a isotypes were measured by ELISA.riefly, 96-well plates (Nunc) were sensitized with rhsp65 protein250 ng/100-�L well) overnight at 4 ◦C. Plates were blocked with:100 dilution of mouse serum for 2 h at 37 ◦C. After washing withBS 0.05% Tween 20, peroxidase-labeled antibodies specific forouse IgG1 or IgG2a isotypes (Sigma) were diluted to 1:5000 and

dded for 2 h at 37 ◦C. The plates were washed seven times with PBS.05% Tween 20 and incubated with H2O2 and o-phenylenediamineor reaction development. Reactions were stopped by the additionf 20 �L of H2SO4, 2N. Optical densities were determinate at 490 nmn an ELISA reader (BioRad, model 2550, CA, USA).

.6. Lymphoproliferation and cytokine production assays

T-cell proliferative responses to Concanavalin-A (ConA) weretudied by [3H]-thymidine incorporation as previously described

27]. Spleen cells were disrupted in RPMI 1640 (Sigma) thatad been supplemented with 2 mM l-glutamine, 1 mM sodiumyruvate, 5% non-essential amino acids (Sigma), 2 ME, strep-omycin (100 �g/mL), and 5% FBS. Cells were washed twice inerum-free RPMI, counted, added to 96-well plates at a cell density

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f 3 × 105 cells/well and subsequently stimulated with ConA4 �g/mL, Sigma). The experiments were performed in triplicatet a final volume of 200 �L/well. After 48 h of incubation at 37 ◦Cnder 5% CO2, cultures were pulsed for 12 h with 1 �Cie/well ofith H3-labeled thymidine. (Amersham, Arlington Heights, IL)

nd then harvested. Incorporation of H3-thymidine was mea-ured using a liquid scintillation counter (Beckman Instruments);ata were expressed as means ± S.E.M. of counts per minute of3-thymidine incorporation.

Supernatants of spleen cells cultured from experimental groupsere used to perform cytokine production assays. The cytokines

nterleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-12 (IL-12),nd interferon-gamma (IFN-�) were measured using a commer-ial ELISA (enzyme linked immunosorbent assay) kit (accordingo the guidelines established by BD Biosciences, San Diego, CA).he absolute cytokine level present in the supernatant preparationsbtained from spleen cell culture was calculated based on a stan-ard curve provided by the commercial kit. Data were expressed ashe mean (log10) ± S.E.M.

.7. Nitric oxide (NO) production assays

The concentration of nitrite (NO2−) in spleen cell supernatants

as measured by a microplate Griess assay as described previously28]. NO2 concentration in culture supernatants was used as anndicator of NO generation and measured with the Griess reagent1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochlo-ide, 2.5% H3PO4). Briefly, 100 �L of the culture supernatants wasdded to an equal volume (v/v) of Griess reagent and incubationas at room temperature for 10 min. The absorbance at 540 nmas determined with a microplate reader. The NO2-concentrationas determined using a standard curve of 1–200 �M of NaNO2.

he conversion of absorbance to �M of NO was deduced fromstandard curve by using a known concentration of NaNO2

iluted in RPMI medium. All determinations were performed inriplicate and expressed with mean ± S.E.M. of �M of NO2. Theerum nitrate concentration was determined by reducing nitrateo nitrite enzymatically using nitrate reductase as described pre-iously [29]. The total amount of nitrite was then determinedsing the Griess method. The results are reported as �M ofO3.

.8. Experimental reproducibility and statistical analysis

All experiments were repeated three times independently. Datarom the different experimental groups were compared using one-ay ANOVA test and multiple comparisons with Dunnet’s post-test.ll calculations and plotting were done using PRISM 5.0 (Graph Padoftware for Science, San Diego, CA, USA). P values were consideredignificant at P < 0.05. All values are means ± S.E.M.

. Results

.1. Pulmonary histopathological and CFU in lungs frommmunized animals with DNAhsp65 and infected with P.rasiliensis

The pulmonary histological examination of the animals fromaline and pVAX1 groups revealed progression of the infection pre-

ominantly in the lungs, characterized by a diffuse inflammatoryesponse. After 30 days of infection, we observed a significant pres-nce of P. brasiliensis in the lung tissue of animals from the infectednd pVAX1 groups. In addition, the lung tissue showed granuloma-ous lesions, which is the characteristic of a P. brasiliensis infection.

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27 (2009) 606–613

e observed a high number of yeast cells in the granulomatousesions with an important inflammatory process. The histopatho-ogical examination of lungs from the DNAhsp65 group 30 daysfter infection revealed small granulomatous lesions with few fun-al cells circumscribed by the granuloma (data not shown).

After 60 days of infection, the inflammatory response hadecome clearly circumscribed, with a clear establishment of gran-lomatous lesions. There was a predominance of epithelioid cells,

imiting the majority of the structures of yeast fungus, whichhowed a proliferative profile with the progression of the infectionFig. 1A and C). There was also the presence of large areas of lungemodeling with extensive deposition of collagen (Fig. 1E). In con-rast, the DNAhsp65 group presented with an inflammatory processf lesser intensity with more defined granolumatous structuresFig. 1B), a clear reduction of yeast cells, and very few depositionsf collagen (Fig. 1D and F).

The reduction of yeast cells observed by histological analysisas confirmed through the P. brasiliensis CFU determination. Fig. 2

hows that the pVAX1 group of animals showed pulmonary fungalurden similar to the saline group after 30 days of infection, with an

ncrease in both groups at day 60. On the other hand, CFU recoveryrom DNAhsp65 mice were nearly half from that observed in thealine and pVAX1 groups at day 30 and even lower at day 60.

The number of CFU after 30 and 60 days of infection in the liverf animals in the DNAhsp65 group was lower than that observed inhe saline group. The same was true for the spleen of immunizednimals relative to the saline group (data not shown).

Taken together, these results indicated that the DNAhsp65 vac-ination confers protection to mice against subsequent infection of. brasiliensis with reduction of lung, liver and spleen injury up tot least 60 days post-challenge.

.2. Humoral immune response of mice immunized withNAhsp65 and infected with P. brasiliensis

Since DNAhsp65 vaccinated animals showed reduction of lungnjury and fungal burdens, we next tried to characterize the mecha-isms responsible for these protective effects. We first determined

f DNAhsp65 vaccination was able to prime the immune system ofice. Thus, we first determined the levels of IgG1 and IgG2a anti-sp65-specific antibodies in serum of the experimental groups 60ays after P. brasiliensis infection (Fig. 3). This analysis revealed noignificant bias towards either increased levels of IgG1 or IgG2a;owever, anti-HSP65 antibodies were detected in the serum of mice

rom the infected and pVAX1 groups at significantly higher levelshan mice immunized with DNAhsp65. These results indicate thatnoculation of the DNAhsp65 vaccine was successful and HSP65rotein was expressed and able to prime the immune system of

mmunized mice. In addition, the observed differences in antibodyroduction suggest that immunization with the DNAhsp65 vac-ine modulates immune response in the animal upon P. brasiliensisnfection.

.3. Effect of DNAhsp65 vaccination on lymphoproliferation andytokine release

In order to determine if the immune response of vaccinated ani-als was modulated we examined cell proliferation and cytokine

roduction. The lymphoproliferation assays using spleenocytesre shown in Fig. 4. After 30 days of infection, mice from the

nfected and pVAX1 groups displayed a noticeable reductionn spleenocyte proliferation in the presence of RPMI medium8191.5 ± 277.9 and 7358.1 ± 412.9 cpm, respectively) and ConA11,226 ± 484.78 and 10250.1 ± 166.7 cpm, respectively) comparedith the control group (17,079 ± 1867 and 30,080 ± 635.6 cpm,

A.M. Ribeiro et al. / Vaccine 27 (2009) 606–613 609

Fig. 1. Histopathological analyses of animals from the pVAX1 and DNAhsp65 groups after 60 days of infection 106 cells of P. brasiliensis. (A) HE, animal from the pVAX1 group(200×). (B) HE, animal from the DNAhps65 group (400×). (C) Silver, animal from the pVAX1 group (400×). (D) Silver, animal from the DNAhsp65 group (400×). (E) Masson(collagen), animal from the pVAX1 group (200×). (F) Masson (collagen), animal from the DNAhsp65 group (200×).

Fig. 2. Fungal burden recovery from lungs of immunized and infected mice with 106

cells of P. brasiliensis. The bars depict means ± S.E.M. (standard error of the mean)of CFU (colony-forming units) obtained from three experimental groups (saline,pVAX1, and DNAhsp65) at 30 d (30 days, n = 10 per group) and 60 d (60 days, n = 10per group) after P. brasiliensis infection. Data is representative of a typical exper-iment that was independently repeated three times. Results are expressed as themean ± S.E.M. (n = 10). *P < 0.05 in relation to saline group.

Fig. 3. IgG2a and IgG1 isotype levels in serum of immunized and infected mice with106 cells of P. brasiliensis. The bars depict levels of anti-rhsp65 IgG2a (open bars) andIgG1 (solid bars) isotypes ± S.E.M. (standard error of the mean) from experimentalgroups (control, saline, pVAX1 and DNAhsp65) at 30 days and 60 days of infection(30 d, 60 d, n = 10). The data represent a typical experiment, which has been repeatedthree times with similar results. *P < 0.05 in relation to saline group.

610 A.M. Ribeiro et al. / Vaccine 27 (2009) 606–613

Fig. 4. Lymphoproliferation of cells in response to stimulation with concanavalin-A(ConA). Spleenocytes were isolated from the experimental groups (control, saline,pVAX1, and DNAhsp65) after 30 days and 60 days of infection (30 d, 60 d, n = 10per group) and cultured in the presence of RPMI medium or ConA (4 �g/mL). Thebars depict means ± S.E.M. (standard error of the mean) of counts per minute of H3-thymidine incorporation by spleenocytes from the experimental groups. The openbars correspond to non-stimulated spleen cells (RPMI), and the solid bars correspondthg

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Fig. 6. NO production in spleen cell culture (NO2) and serum (NO3) from mice immu-nized and infected with 106 cells of P. brasiliensis. The bars depict means ± S.E.M. ofNO production by cell culture (NO2) and serum (NO3) from experimental groups(control, saline, pVAX1 and DNAhsp65) at 30 days and 60 days after infection (30 d,6tr

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on-stimulated and stimulated, respectively). Spleenocyte prolif-ration levels of mice from the DNAhsp65 group (15444.8 ± 771.1nd 27755.8 ± 776.8 cpm, non-stimulated and stimulated, respec-ively) returned to values similar to those observed in the controlroup. We found similar results after 60 days of infection. Fig. 4hows such restoration in animals immunized with DNAhsp65fter 60 days of P. brasiliensis infection.

We next studied the culture supernatants of spleenocytes fromxperimental groups to evaluate the release of various cytokines

ncluding: IL-4, IL-10, IL-12, and IFN-� (Fig. 5). We found, that after0 days of infection, mice immunized with DNAhsp65 producedignificantly higher levels of IFN-� (6207.3 ± 61.2 pg/mL) and IL-123370 ± 259.6 pg/mL) and similar levels of IL-4 (6251.3 ± 141 pg/mL)nd IL-10 (150 ± 23 pg/mL) in comparison to levels detected in the

ig. 5. Levels of IL-4, IL-10, IL-12, and IFN-� produced by spleenocytes from mice immunizf cytokine production (pg/mL) by spleenocytes from mice from the experimental groups, 60 d, n = 10). The data represent a typical experiment that has been repeated three tim

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aline mice. Cytokine productions of saline and pVAX1 groups wereimilar to those of the control group. A similar profile was foundfter 60 days of infection. As shown in Fig. 5, the group immunizedith DNAhsp65, 30 and 60 days after infection, had higher levels

f IFN-� and IL-12 compared to the saline group, and there was noifference in IL-4 and IL-10. The results were expressed as the meanlog10) ± S.E.M.

As a control for cytokine production, we evaluated the immuneesponse in mice immunized with 3 doses of the DNAhsp65accine that were not infected. These animals displayed a polar-zation/modulation towards the Th1-type immune response (data

ed and infected with 106 cells of P. brasiliensis. The bars depict means (log10) ± S.E.M.(control, saline, pVAX1, and DNAhsp65) at 30 days and 60 days after infection (30

es with similar results. Results are expressed as the mean (log10) ± S.E.M. *P < 0.05

ot shown).Therefore, the data from humoral and cellular immunity showed

hat DNAhsp65 vaccination primed the immune system for pro-uction of specific antibodies by the modulation of the immuneesponse.

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.4. NO3 and NO2 production after immunization and infectionith P. brasiliensis

Finally, considering that one of the most important fungicidalechanisms is mediated by nitric oxide (NO), we evaluated the pro-

uction of NO through the production of NO3 and NO2 in the serumnd supernatant from spleen cell cultures, respectively (Fig. 6). Theevels of NO3 and NO2 were measured after 30 and 60 days of P.rasiliensis infection. After 30 days of infection, the animals fromhe infected and pVAX1 groups presented with similar amounts ofO3 (12 ± 1.1 and 11.3 ± 0.66 �m, respectively) and NO2 (11.3 ± 0.3nd 13.6 ± 1.2 �mol/L, respectively) compared to the control group11.6 ± 0.8 �m and 9.3 ± 0.8 �mol/L, NO3 and NO2, respectively).owever, the immunized animals with DNAhsp65 showed highmounts of both NO3 (57 ± 3 �m) and NO2 (108 ± 2.5 �m/L) relativeo the control group of mice. After 60 days of infection, mice immu-ized with DNAhsp65 showed sustained NO3 and NO2 productionFig. 6).

Taken together, these data suggest that DNAhsp65 vaccinations able to prime the immune response of mice and can protectgainst a subsequent infection with P. brasiliensis with fungal lysesediated by NO.

. Discussion

While the incidence of fungal infections has increased world-ide, the use of standard anti-fungal therapies is generally

imited because of toxicity, low efficacy rates, and drug resis-ance. Therefore, success rates for the treatment of many mycosesemain unacceptably low. To tackle these difficulties, alternativepproaches to the prevention and treatment of mycoses need to beeveloped. Vaccination has been one of the most important inter-entions designed to prevent diseases worldwide. The vaccinationpproach requires the selection of immunogenic components thatead to protection. Novel approaches require a much more detailedharacterization of the genetic organization of the pathogen andhe genes responsible for virulence or induction of immunity. Withegard to fungal diseases, these genes have been investigated inistoplasma capsulatum (H. capsulatum), P. brasiliensis, Blastomycesermatitidis, Cryptococcus neoformans, Phythium insidiosum, andoccidioides immintis [30]. HSPs are also known to be immunogenicroteins, and vaccines containing either HSPs genes or the pro-eins themselves have been tested against human fungal diseases,ncluding mycoses caused by Candida spp. [31] and H. capsulatum32,33]. In murine models with invasive candidiasis, antibody pro-ection can occur through recognition of specific proteins, such asSP90 [31]. In experimental models, the recombinant heat-shockrotein 60 (rHSP60) induces resistance in immunocompetent ani-als challenged with H. capsulatum. Specifically, CD4+ T cells and

h1 cytokines are required for this resistance [32,33]. Experimental34,35] and clinical [36,37] investigations have indicated that cellu-ar rather than humoral immunity is the most effective host defense

echanism that controls the pathogenesis of Paracoccidioidomy-osis. Specific cell-mediated immune responses seem to play anmportant role in resistance to P. brasiliensis. Patients with systemicaracoccidioidomycosis tend to show depressed cellular immuneesponses compared to those with localized disease [36]. Also, theost severe forms of infection are associated with high levels of

pecific antibodies [reviewed in 38]. In the murine model, resis-ant mice are assumed to direct their immune response towards a

referential T helper cell type 1 (Th1) activation with production of

FN-�, and efficient macrophage activation is able to contain fun-al dissemination and disease progression [39,40]. In addition, thistudy demonstrated for the first time that IFN-� exerts a protectiveole against pulmonary P. brasiliensis infection irrespective of the

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27 (2009) 606–613 611

enetic background of the mouse strain. IFN-� depletion increasedhe pulmonary fungal burden of B10 mice more than 1000-foldnd increased the already high number of fungal cells in the lungsf A/Sn mice 10-fold [41]. Souto et al. [42] used mice geneticallyeficient in IFN-� or TNF-� receptor p55 and demonstrated thatoth cytokines are involved in resistance to P. brasiliensis infection,ranuloma formation, and controlling fungus dissemination. PCMn mice genetically deficient in IL-4 was less severe than in normal

ice and was associated with an impaired Th2 immune response43].

The immunoprotective role of the major diagnostic antigen ofaracoccidioidomycosis, the Gp43 protein [44,45], was also inves-igated. Pinto et al. demonstrated that BALB/c mice immunizedith the gp43 gene from P. brasiliensis evoked a cell-mediated

mmune response. The P. brasiliensis DNA-based vaccination usinghe gp43 gene induced a specific, long-lasting, mixed Th1/Th2 cel-ular immune response [46]. More recently, immunization withhe peptide P10 (derived from gp43) and chemotherapy weresed together (as adjuvant) in an attempt to improve treatmentf Paracoccidioidomycosis and to prevent relapse. The combinedreatment showed an additive protective effect [47].

In this context, knowing the beneficial effects of the M. lepraesp65 genetic vaccine against diseases such as tuberculosis [12–15],rthritis [17] and leishmaniasis [16], we decided to test it againstaracoccidioidomycosis. In this study, we reported for the first timehe efficacy of the DNAhsp65 immunization against a fungal infec-ion, Paracoccidioidomycosis.

Similar to the results obtained with the gp43 vaccine [46], ani-als immunized with the DNAhsp65 vaccine showed reduced

ulmonary fungal burden as well as increased levels of IFN-�. Inoth studies, the fungi were not eliminated, but their presence wasignificantly reduced. Furthermore, histopathological analyses ofhe lungs revealed fewer fungal cells and deposition of collagen inhis organ, both elements accounting for decreased loss of function.

The animals immunized with the hsp65 gene presented anncrease of Th1 immune response. This was in contrast with thep43 DNA-vaccine, which led to a more pronounced productionf IgG1 with low levels of IgG2a and IgG2b, which indicates ah2 response. In animals immunized with DNAhsp65, the levels ofgG2a were significantly higher than those of IgG1. Additionally, webserved an increase in IFN-� production and IL-12 levels, anothermportant cytokine. Romano et al. [48] showed that patientsresent defective lymphoproliferation, IFN-� responses to the main. brasiliensis antigen (Gp43) and synthesis of IL-12, which cor-elates with disease severity. Our results indicate that the micemmunized with DNAhsp65 present elevated IFN-� and IL-12 lev-ls but their IL-4 and IL-10 levels remain unchanged relative tohe saline-treated mice. Therefore, there was an increase of Th1ytokines levels that are central to resistance to this disease.

Another important finding was the elevated concentration ofO in the serum and spleen cell cultures from mice immunizedith DNAhsp65. It is known that NO works as a potent microbicide

actor and that its secretion is related to the elimination of fungusnd protection for the host.

Therefore, the DNAhsp65 vaccination triggered a more con-istent protective response, comprising novel advantages, whenompared to the results obtained with the gp43 DNA vaccine.

The use of plasmid DNA in gene therapy can be safe, althoughntegration into the host cell genome could produce insertional

utagenesis, which would have the potential of activating or inacti-

ating genes. Coelho et al. [16] showed that our vaccine (DNAhsp65)s reliable for gene therapy as well as for vaccination in a clinicaletting because it was not integrated into the mouse genome. Ourndings are promising in the context of new systemic mycosis vac-ination strategies. The DNAhsp65 vaccine holds a lot of promise

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n the field of mycology; therefore, it may be an attractive candi-ate for prevention, therapy, and an adjuvant for mycosis treatment.urrently, however, there are no studies that describe the utility ofNA vaccination involving hsp65 gene and fungal infections. The

esults presented here suggest that the DNAhsp65 vaccine coulde explored for other systemic mycoses, opening new perspectives

n immunization/prevention (immunoprophylaxis) and treatmentimmunotherapy). We should bear in mind that higher dosagesf vaccine are required when considering naked DNA because thextracellular delivery of DNA results in degradation of the hsp65ene by nucleases within the muscle tissue following conven-ional administration [49]. Thus, other methods of delivery haveeen developed for candidate DNA vaccines. The encapsulation ofNA into biodegradable microspheres such as those based on poly

lactic-co-glycolic acid) copolymers (PLGA) provides an effectiveay to protect the DNA against biological degradation by nucleases

nd permits a continuous release of DNA in a controlled mannerver a period of time [50,51].

With regards to the development of novel pharmaceutical for-ulations utilizing the DNAhsp65, an array of products based on

ontrolled-release drug delivery systems (e.g., PLGA microspheresnd liposomes) is already available. These systems meet the chal-enges faced by the development pipeline of vaccines with potential

orldwide impact. In fact, recent results from our group havehown that the DNAhsp65 can be reduced 16 times when lipo-omes are used as a delivery system in the murine model of annti-tuberculosis vaccination, which would retain the prophylacticffects obtained with naked DNA [52].

Finally, here we demonstrated the successful vaccination against. brasiliensis infection, not only reducing fungal burden by driv-ng the immune response towards a Th1 immune response and NOroduction, but also reducing lung remodeling, an aspect of utmost

mportance due to the high grade of lung remodeling observed inisease progression. All these data showed that Hsp65 has potentials an immunomodulator and demands the discovery of the molecu-ar mechanisms behind how this protein mediates the prophylacticnd therapeutic effects in different illnesses.

cknowledgements

We are grateful to Izaíra Tincani Brandão, Ana Masson andiviane Monteiro Leal for technical assistance. This work was sup-orted by a grant from Coordenacão de Aperfeicoamento de Pessoale Nível Superior (CAPES) and Conselho Nacional de Desenvolvi-ento Científico e Tecnológico (CNPq).

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