DNA-based vaccination against murine paracoccidioidomycosis using the gp43 gene from Paracoccidioides brasiliensis

Vaccine 18 (2000) 3050±3058

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DNA-based vaccination against murine paracoccidioidomycosis using the gp43 gene from Paracoccidioides brasiliensis Aguinaldo R. Pinto a, c, Rosana Puccia a, Susana N. Diniz d, Marcelo F. Franco b, Luiz R. Travassos a,* a

Disciplina de Biologia Celular, Universidade Federal de SaÄo Paulo, Rua Botucatu 862, 88 andar, SaÄo Paulo, SP 04023-062, Brazil b Departamento de Patologia, Universidade Federal de SaÄo Paulo, Rua Botucatu 740, SaÄo Paulo, SP 04023-062, Brazil c Sec° aÄo de Sorologia, Instituto Adolfo Lutz, Av. Dr. Arnaldo 351, 108 andar, SaÄo Paulo, SP 01246-902, Brazil d Departamento de BioquõÂmica e Imunologia Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil Received 25 August 1999; received in revised form 7 February 2000; accepted 15 February 2000

Abstract Gp43, the major 43-kDa antigenic glycoprotein of Paracoccidioides brasiliensis, or its 15-amino acid inner peptide (P10), induces a T-CD4+, Th1 cellular immune response which protects BALB/c mice from intratracheal infection by virulent yeast forms. We investigated whether DNA vaccination using the gp43 gene could elicit protective immunity against P. brasiliensis. Animals immunised intramuscularly (i.m.) or intradermally (i.d.) with plasmid DNA containing the gp43 gene induced a speci®c, long lasting humoral and cellular immune response. A mixed Th1/Th2 cellular immune response in DNA-immunized mice was modulated in vivo by IFN-g and was protective in BALB/c mice. A signi®cant decrease in the lung colony forming units (CFUs) and reduced, or no dissemination to the spleen and liver of immunised mice were observed. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Paracoccidioides brasiliensis; DNA immunisation; gp43

1. Introduction Paracoccidioides brasiliensis is a dimorphic fungus which causes paracoccidioidomycosis (PCM), a systemic disease that a€ects mostly rural workers but can also reach urban centers upon migration of infected individuals. It is widespread in Latin America, with endemic areas extending from Central America to Argentina, and it is the prevalent deep mycosis in this region [1]. Primary infection is usually acquired via inhalation of propagules that give rise to infective yeast forms in the host tissue. Mostly asymptomatic, PCM may evolve in as many as 2% of all infected individuals estimated as 10 million [2]. The acute and * Corresponding author. Tel.: +55-11-576-4523; fax: +55-11-57115877. E-mail address: [email protected] (L.R. Travassos).

subacute forms of PCM predominate in young people of both sexes, mainly a€ecting the lymphatic system. The chronic form has a greater incidence in adult males with the lungs being most frequently a€ected, with or without mucocutaneous involvement. Experimental [3,4] and clinical [5,6] evidence indicates that cellular rather than humoral immunity is the e€ective host defense mechanism that controls pathogenesis and evolution of PCM. A correlation has been found between the severity of the disease and impaired DTH (delayed-type hypersensitivity) [5], which is a consequence of the anergic state. Furthermore, severe forms of PCM are associated with high levels of speci®c antibodies [7] and hypergammaglobulinemia which, however, fail to protect against the disease. The vaccination approach to prevent widespread infectious diseases requires the selection of immunogenic components that lead to protection. In the case

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of fungal diseases these molecules have been investigated in Histoplasma capsulatum, P. brasiliensis, Blastomyces dermatitidis, Cryptococcus neoformans, Phythium insidiosum [8] and recently, Coccidioides immitis [9]. In P. brasiliensis, the immunoprotective role of the major diagnostic antigen, the 43 kDa glycoprotein [10,11], is currently being investigated in our laboratory. The gp43 is continuously secreted by growing yeast forms [12], reacts with virtually all sera from infected patients [13,14], and contains epitopes which elicit a cellular immune response (DTH) in both guinea pigs [15] and human patients [16]. Mice sensitised with the gp43 in CFA produce speci®c T-CD4+ but not TCD8+ cells [17]. These reactive T-CD4+ cells are of the Th1 subtype, producing interferon-g and IL-2, but not IL-4 or IL-5. Gp43-reactive T lymphocytes are also stimulated by a 15-amino-acid peptide (P10) contained in the gp43 which carries the main epitope inducing T-cell proliferation and protection against PCM in BALB/c mice [18]. The gp43 gene has been cloned and completely sequenced in our laboratory [19]. This antigen may also act as a virulence factor, since it binds speci®cally and with high anity to murine laminin-1. Laminin-coated yeasts have much enhanced virulence in a hamster-testicle infection model [20] and this e€ect is abrogated by anti-gp43 monoclonal antibodies [21]. DNA vaccines have been used against bacterial [22], virus [23] and protozoan [24] infections and also against tumor cells [25] with various eciencies. Direct inoculation of the gene promotes synthesis by the host of speci®c foreign proteins, which become targets of the immune surveillance mechanisms [26,27]. Since the native gp43 generates a protective immune response in mice against a challenge by virulent P. brasiliensis [18], we have presently investigated the immune response and protection by a plasmid expressing the gp43 gene under control of the CMV promoter. The results clearly show that DNA vaccination elicits speci®c immune responses, which are protective against highly infective yeast forms of P. brasiliensis. 2. Materials and methods 2.1. Plasmid construction VR1012 vector (kindly provided by Vical Inc., San Diego, CA, USA [28]) was used for the expression of the gp43 peptide sequence in mice. cDNA of the gp43 antigen was obtained by RT±PCR (reverse transcriptase polymerase chain reaction), using total RNA isolated from P. brasiliensis [19] and primer 511 (5 '-GATCCGAGTCGACATCGTTTTTTTTTTTTTTTTT-3 ') to synthesize total cDNA. The reaction (20 ml ®nal) was carried out in 50 mM Tris±HCl pH 7.0, con-

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taining 50 mM KCl, 10 mM MgCl2, 10 U of RNAsin (Promega Biotech), primer 511 (500 ng), 10 U of AMV reverse transcriptase (Pharmacia Biotech Inc.) and dNTPs (1 mM of each). Total RNA (5 mg) was heated for 3 min at 658C and added to the mixture, which was ®rst incubated at 428C for 1 h and then at 528C for 30 min. The cDNA was precipitated with ethanol, diluted in 1 ml of Tris±EDTA bu€er (TE), and used as template in the PCR reactions. The full coding sequence of the gp43 was synthesized by using the upstream primer 490 (5 '-GTCAGATCTATCATGAATTTTAGTTCCTTAAC-3 '), containing a Bgl II site before the ATG start codon, and the downstream primer 491 (5 '-ACGTCGACTCACCTGCATCCACCATACTT-3'), containing a Sal I site immediately after the TGA stop codon. The PCR reactions (100 ml) were carried out following the basic protocol provided by Perkin Elmer, using total cDNA (30 ml) and 1 mM of each primer. The reactions started with one cycle of 948C (5 min), 558C (2 min) and 728C (40 min), for the synthesis of double stranded cDNA, followed by 30 cycles of 948C (30 sec), 558C (30 sec) and 728C (2 min), and a ®nal 10-min extension at 728C. The PCR fragment generated with primers 490/491 (1250 bp) migrated faster in agarose gel electrophoresis than the control fragment obtained in the same conditions using genomic DNA as template. The di€erence in migration was compatible with the absence of the 78-bp intron [19]. The ampli®ed cDNA bands were extracted from the agarose gels using the SephaglassTM kit (Pharmacia Biotech Inc.), cloned in the pMOS T Blue vector (Amersham), excised by Bgl II and Xba I restriction and sub-cloned in the VR1012 by directional insertion in the Sal I/Xba I sites, using a blunt ligation between the Bgl II and the Sal I ends. The resulting plasmid was called VR-gp43 and was puri®ed in large scale by caesium chloride density gradients according to standard protocols [29]. The parental plasmid VR1012 was prepared in the same way, to be used as a control. Plasmid concentration was determined by optical density at 260 nm and semiquantitatively by agarose gel electrophoresis relative to a standard. Puri®ed preparations were stored in phosphate bu€ered saline pH 7.4 (PBS) at ÿ208C until use. Expression of the gp43 in vitro was obtained in COS-7 cells, which were transiently transfected with VR-gp43 and VR1012 using Lipofectin (Life Technologies), according to the protocol provided by the manufacturer. After 24 h in culture, total mRNA of COS-7 cells was puri®ed, reverse-transcribed (First-Strand cDNA Synthesis kit Ð Pharmacia) and the cDNA ampli®ed by PCR using the 490/491 primer pair, in 35 ampli®cation cycles of 948C (1 min), 608C (1 min) and 728C (3 min). PCR products were visualised in 1% agarose gels stained with ethidium bromide.

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2.2. Puri®cation of the gp43 The gp43 was puri®ed from culture ®ltrates of P. brasiliensis, strain 339 (originally obtained from A. Restrepo-Moreno, Medellin, Colombia), by anity chromatography in columns with anti-gp43 17C monoclonal antibody, as previously described [30]. The protein content of the puri®ed preparations was determined by Bradford's method [31]. 2.3. Immunisation of mice with plasmid DNA Groups of 6±8-week-old female BALB/c mice were immunised either intradermally (i.d.) at the tail base or intramuscularly (i.m.) in both quadriceps with four doses of 100 mg of VR-gp43 at 2-week intervals. Serum samples were analysed for the presence of anti-gp43 antibodies 2 weeks after each immunisation. Control groups were immunised with the parental plasmid VR1012 following the same schedule. 2.4. Antibody response to the gp43 Anti-gp43 antibodies were detected by conventional ELISA [11] using microtitre polystyrene plates (Nunc, MaxiSorpe surface) coated with 250 ng/well of puri®ed gp43 and 10% dehydrated skim milk in phosphate bu€ered saline (PBS) for blocking. Serum pools from immunised and control mice were tested by serial dilution starting at 1:50 and quanti®ed with an antimouse Ig±biotin±streptavidin±peroxidase system (Sigma Chemical Co). The results were recorded as A492 readings taken in a Titertek Multiskan MCC/340. Immunoglobulin classes and subclasses were determined with the Mouse Typer Sub-isotyping Kit (BIORAD) from serum pools diluted 1:200. The speci®city of the antibody response was further evaluated by immunoblotting. Culture ®ltrates of P. brasiliensis grown at 358C were separated in 10% SDS±PAGE gels, transferred to a nitrocellulose membrane [32] and tested with mouse serum samples diluted 1:2000. The reaction was revealed with anti-murine Ig peroxidase conjugate by chemiluminescence. A mouse anti-gp43 polyclonal anti-serum and a pool of mouse sera immunised with VR1012 were used as positive and negative controls, respectively. 2.5. T-cell proliferative response and lymphokine assays The T-cell proliferation assays were carried out with inguinal and popliteal lymph node (LN) cells obtained from mice immunised with four doses of plasmid and CFA (50 ml emulsi®ed in PBS) injected in the hind foot-pad concomitantly with the fourth dose of the plasmid. The cells were washed twice and the pellet suspended in RPMI 1640 (Life Technologies) sup-

plemented with 20 mM NaHCO3, 10 mM HEPES, 100 U penicillin/ml, 100 mg of streptomycin/ml, 2 mM Lglutamine, 50 mM b-mercaptoethanol, 5 mM sodium pyruvate, 100 mM nonessential amino acids and 1% (v/v) normal human serum (complete culture medium). The cells were counted in 0.1% Trypan blue and the viable cells were cultured at a density of 4  105 cells/ well in 96-well Costar plates (0.2 ml/well), without antigen (negative control) or with 1±50 mg/ml of gp43 or 2.5 mg/ml of Concanavalin A. Cultures were incubated for 4 days at 378C under 5% CO2. In the last 18±20 h, 1 mCi of [3H]-thymidine (Amersham) was added to each well. Proliferation was determined by incorporation of radioactivity, and the results (in counts per minute) are shown as means of triplicate determination2standard deviation. A separate control group of animals was inoculated subcutaneously with native gp43 (a single dose of 50 mg in complete Freund's adjuvant) and the LN cells from these animals were used as positive controls of the T-cell proliferative response. Lymphokine release assays were conducted in 24well plates by culturing 1  107 LN cells in 1 ml of complete culture medium without or with 25 mg/ml of gp43 for 48 h. The culture supernatants were collected, separated from the cell debris and stored at ÿ708C. The presence of gamma-interferon (IFN-g), IL-4, IL-5, IL-10, and IL-12 was analysed by sandwich ELISA using antibody pairs purchased from Pharmingen (San Diego, CA, USA). The ELISA procedure was performed according to the manufacturer's protocol, except that the substrate used was orthophenylenediamine (Sigma) at 500 mg/ml, and the reactions were read at 492 nm. Recombinant cytokines (Pharmingen) were used for standard curves with the respective monoclonal antibodies. For IL-2 detection, the supernatants were added to 1  104 cells/well of an IL-2respoding murine tumor-speci®c cytotoxic T-lymphocyte line (CTLL) [33]. Brie¯y, the cells were incubated for 30 h with 0.5 mCi of [3H]-thymidine added 6 h before collecting the cells on a ®berglass ®lter for radioactivity counting. The results were expressed as the averages of four determinations. Negative controls were run with the RPMI medium alone or the supernatant of a LN cell culture without gp43. The positive control consisted of CTLL cells incubated with rIL-2 at 10 U/ml. 2.6. Protection assays Three weeks after the last dose of plasmid DNA, each mouse was inoculated intratracheally with 2  105 yeast forms of virulent P. brasiliensis strain 1914 grown in YPD-agar and suspended in sterile PBS. A maximal volume of 50 ml was inoculated per mouse. The number of viable microorganisms in di€erent

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organs in infected mice was determined by the number of colony-forming units (CFU). After 3 months of infection, mice were sacri®ced and their lungs, spleens, and livers were removed, weighed, homogenized, and washed three times in PBS by centrifugation. The ®nal suspension in PBS was plated on brain heart infusion agar supplemented with 4% fetal calf serum and 5% P. brasiliensis spent culture medium as growth factor. Gentamicin and cycloheximide were added at 40 and 500 mg/l, respectively. The plates were incubated at 368C for 20 days. The results were expressed as the number (log10) of viable P. brasiliensis per gram of tissue per mouse. 2.7. Histopathology Groups of BALB/c mice immunised with VR-gp43 or VR1012 were infected intratracheally and killed after 3 months. The lungs, spleens, and livers were excised, ®xed in 10% bu€ered formalin, and embedded in paran for sectioning. The sections were stained with hematoxylin-eosin and examined microscopically (Optiphot-2; Nikon, Tokyo, Japan). 3. Results The expression of P. brasiliensis gp43 in mammalian cells was accomplished using the VR1012 vector (Vical Inc.) carrying the human cytomegalovirus (CMV) promoter, bovine growth-hormone (BGH) terminator and the kanamycin resistance marker. The insert used in the VR-gp43 construct contained the antigen ORF including the signal sequence. In order to ensure that the plasmid DNA was intact and functional, COS-7 cells were transiently transfected with either VR-gp43 or the parental plasmid (VR1012). At 24 h after transfection, the COS-7 mRNA was extracted, reverse-transcribed and ampli®ed by PCR with the gp43 speci®c primers. As shown in Fig. 1, the expression of the

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Fig. 1. Ethidium bromide-stained agarose gel showing the RT±PCR analysis of COS-7 cells transiently transfected with either the VRgp43 (lane 3) or VR1012 plasmid (lane 1). Lane 2 is the PCR product using genomic DNA of P. brasiliensis as template (positive control). Migration of the 1.3 kb marker is indicated.

gp43 as speci®c cDNA (01.3 kb) was obtained in cells transfected with VR-gp43 (lane 3), but not with VR1012 (lane 1). Controls without the reverse transcriptase were not ampli®ed showing that the amplicon seen in lane 3 was a transcription product. The ecacy of the i.d. and i.m. immunisation in mice with the VR-gp43 plasmid construct was determined by the antibody response against the gp43 in ELISA tests. As seen in Fig. 2a, with either inoculation route the humoral response increased after each booster and the maximal antibody production was detected after the fourth dose. Since higher antibody titres were obtained with the i.m. immunisation, this injection route was used in the subsequent experiments. Although a comparison is dicult to make because the delivery of the immunogen into the immunised animal di€ers in time and amount, the antibody production in BALB/c mice was higher with a single subcutaneous (s.c.) injection of the gp43 than after four doses of the VR-gp43 (Fig. 2b) over a 2-month period. The humoral response was long lasting in the DNA-immunised mice. The antibody titres in sera of animals tested 6 months and 15 days after the last immunisation dose were similar (data not shown). In the VR-gp43-immunised mice, IgG1 antibodies predomi-

Table 1 Cytokine production in lymphocyte cultures from mice immunised with the VR-gp43 after 60 days of the onset of immunisationa

Medium SC SC+gp43 SC+ConA

IL-2 (cpm.10ÿ3)

IL-4 (pg/ml)

IL-5 (pg/ml)

IL-10 (pg/ml)

IL-12 (pg/ml)

IFN-g (U/ml)

0.420.1 2.921.1 16.821.2 1320.5

±b ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± 650c 690c

a Cells were cultured for 48 h with 25 mg of gp43/ml, 2.5 mg/ml Concanavalin A or without stimulus (SC=supernatant of culture). IFN-g, IL4, IL-5, IL-10, and IL-12 were analysed by capture enzyme-linked immunosorbent assay (ELISA) using Pharmingen antibodies. IL-2 in the culture supernatant was detected with a tumour-speci®c cytotoxic T-lymphocyte line (CTLL). Values are average of four determinations. Lymphocytes cultured in presence of ConA incorporated 50,000 cpm of 3H-thymidine; 3000 cpm with no stimulation, and 45,000 cpm stimulated with the gp43. CTLL incubated in presence of 10 U/ml of rIL-2 incorporated 20,900 cpm. b ±, not detected. c Values obtained from one representative experiment out of four.

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Fig. 2. Humoral immune response after VR-gp43 inoculation. (a) A serum pool (1:50) from 15 mice inoculated i.m or i.d. with the VR-gp43 was tested for total Ig response against the gp43 by ELISA. Sera were obtained 2 weeks after each DNA inoculation. The results represent the means of triplicates2SD. (b) Antibodies to gp43 in mice immunised with four doses of VR-gp43 (.) or VR-1012 (w), compared with mice immunised with one s.c. dose of gp43 (50 mg) in CFA (Q), the serum collected 2 weeks after immunisation; (q) pre-immune serum. Values are the average of two determinations with pools of ®ve sera for each system (c) Class and sub-class Ig against the gp43 were determined by ELISA with the serum pool collected at day 60 after the start of i.m. immunisation.

nated, although IgG2a, IgG2b, IgG3, and IgE were also present in the sera of immunised animals; in contrast, IgA and IgM antibodies were not detected (Fig. 2c). Control animals immunised with VR1012 did not react with the gp43 (data not shown). The

Fig. 3. Immunoblotting of serum pools (1:2000) from mice injected with the VR1012 (lane A) or VR-gp43 (lane B) against culture ®ltrates from P. brasiliensis. Lane C, control reaction with anti-gp43 mouse polyclonal antibody. The arrowhead indicates the migration position of the gp43.

speci®city of the antibody response was evaluated using a P. brasiliensis culture ®ltrate and immunoblotting with pooled serum samples collected at day 60 from animals immunised i.m. with VR-gp43 and the control plasmid. Results showed that the antibodies generated in the DNA-injected mice were speci®c for the gp43, which was the only component detected in the immunoblot (Fig. 3, lane B). To show the ability of VR-gp43 to elicit a cellular immune response, lymphocytes from the draining lymph nodes were harvested 2 weeks after the last plasmid inoculation and stimulated in vitro with the gp43. Lymphoid cells from mice immunised with VRgp43 proliferated intensely when stimulated with di€erent concentrations of gp43. The proliferation level was comparable with that of LN cells from mice immunised subcutaneously with a single dose of 50 mg of native gp43 in complete Freund's adjuvant (Fig. 4a). The response was speci®c for gp43, since no proliferation was detected with nonrelated proteins, such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Immunisation with VR1012 did not yield LN cells responsive to the gp43 (data not

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Fig. 4. (a) Cellular immune response to gp43 in mice immunised with the VR-g43 plasmid or native gp43. Lymphoid cells were collected 2 weeks after immunisation and stimulated in vitro with di€erent amounts of gp43 for 96 h, as described in Section 2.5. Values are means of triplicate determination2SD. Lymphoid cells cultured with Concavanalin A for 48 h gave values of ca. 60,000 cpm. (b) Cellular immune response was compared 15 days and 6 months after the end of immunisation. The stimulation index was calculated by dividing the mean radioactivity in cpm of cells stimulated by the gp43 (at the indicated concentrations), by that of the control (lymphoid cells cultured in complete medium).

shown). The cellular immune response in mice immunised with VR-gp43 persisted for at least 6 months after the last plasmid dose, although not as intense as that measured 15 days after immunisation (Fig. 4b). Lymphoid cells from mice immunised with VR-gp43 when re-stimulated in vitro with the gp43 produced substantial amounts of interleukin-2 (IL-2) and IFN-g whereas IL-4, IL-5, IL-10, and IL-12 were not detected (Table 1). A similar interleukin pattern was obtained in mice tested 6 months after the last plasmid dose, with a decrease in the IFN-g concentration (data not shown), simultaneously with a decrease in the rate of cell proliferation as seen in Fig. 4b. To determine whether DNA vaccination with the gp43 gene was protective against murine PCM, plasmid-immunised and control mice were challenged by intratracheal injection of 2  105 yeast forms of the virulent P. brasiliensis isolate 1914. Three months after prime infection, mice were killed and the CFUs were determined in the lungs, spleens and livers. Mice immunised with the VR-gp43 showed signi®cant reduction of CFUs in these organs, when compared with animals that received only the VR1012 plasmid (Fig. 5). The histopathological analysis of mice immu-

Fig. 5. Protective e€ect of immunisation with theVR-gp43 plasmid against intratracheal infection by virulent P. brasiliensis 1914. The CFU was estimated as described in Section 2.6. Eight mice were injected with the VR1012 and 10 mice were injected with the VRgp43 plasmid. The individual CFUs are shown with averages as horizontal bars.

nised with VR1012 showed an extensive destruction of the lung tissue, and exudative epithelioid granulomas with numerous viable multiplying fungi. The liver and spleen showed the same exudative lesion with fewer viable fungi. In the VR-gp43 immunised animals, very few and small granulomas in the lungs were seen, with no detectable fungal cells in the lungs, spleen or liver (Fig. 6).

4. Discussion The present data demonstrate that immunisation of BALB/c mice with a mammalian expression vector (VR-gp43) carrying the full gene of the gp43 of P. brasiliensis under the control of CMV promoter induces B and T cell-mediated immune responses against the translated product of this gene. Such immunisation is protective against the intratracheal challenge by virulent P. brasiliensis yeast forms. In PCM, as in other systemic mycoses, cell-mediated immunity is the most relevant defense mechanism. In a murine model [34], the activation of type-1 cellular immune response with production of IFN-g, IL-2, TNF-a and the ecient macrophage activation were able to contain fungal dissemination and the progression of disease. It has been suggested that the protective e€ect of either the 43 kDa glycoprotein or peptide 10 (P10), which contains the T-CD4+ epitope, could be attributed to the in vivo production of IFN-g in the immunised animals [18]. The role of IFN-g for enhanced killing of P. brasiliensis [35] and protection in the same infection model presently used [36] has been determined. Recent evidence [37] showed that mice de®cient in IFN-g receptor were highly susceptible to P. brasiliensis intratracheal infection with increased morbidity and 100% mortality after 5 weeks of infection. In mice de®cient in IFN-g or IFN-g-R, but not IFN-a,b, important dissemination of the infection was observed, with pronounced cachexia. Interest-

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Fig. 6. Representative histopathology of lesions caused by P. brasiliensis. (A [100]) Lung section from a mouse immunised with the VR-gp43, with no detectable fungal cells. (B [100]) Lung from mice immunised with VR-1012 showing extensive granulomatous lesions replacing the normal tissue, with a great number of viable yeast cells.

ingly, animals de®cient in IRF-1 behaved very similarly to the IFN-g-R knock-outs [38]. The immunisation of mice with the VR-gp43 plasmid evoked a cell-mediated immune response, as measured by in vitro proliferation of lymph node cells stimulated with the native gp43 antigen. The amount of IFN-g produced by gp43-stimulated lymphoid cells was comparable to that using a mitogen, indicating a powerful immune response. The plasmid DNA immu-

nisation is therefore able to mount an e€ective, longlasting cellular immune response, mediated by IFN-g, which is protective in mice challenged with a highly virulent strain of P. brasiliensis. Over the past years, DNA immunisation has been intensively studied in animal models aiming at protective immunity against a variety of infections. DNA vaccines provide antigens required for protective immunisation of the host without the need of

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live organisms or replicating vector [39]. This kind of immunisation is unlikely to be harmful to host cells since most plasmids exist in a nonintegrated, circular form and do not replicate [40]. Because the immunogen is synthesized within the host by cells which have taken up the antigen encoding DNA, in vivo protein synthesis allows for processing, modi®cation and presentation of the antigens to the immune system in a way similar to that of the natural infection. In addition, DNA vaccines are able to express their constituent genes for long periods [41] compared to the shorter half-life of recombinant proteins. The present results con®rm that the immune response elicited by the VR-gp43 is also long lasting, with humoral and cellular speci®c responses being e€ective 6 months after the last DNA inoculation. Several routes have been used for the administration of DNA vaccines, namely intramuscular, intradermic, intravenous, intranasal, and gene-gun mediated [42]. We compared the immune e€ectiveness of two routes of administration, i.m. and i.d. The i.m. route rendered higher titers of speci®c gp43 antibodies and was chosen in our experiments. Although the intramuscular injection of plasmid DNA has been used by several groups, muscle cells do not express MHC class II and accessory molecules, such as ICAM-1 and LFA-3, both needed for induction of CD4+ T cells [43]. Transfected muscle cells probably shed the synthesized antigens, which are then processed by APCs, or alternatively, resident and recruited dendritric cells could be transfected upon inoculation of plasmid DNA, leading to activation of the T cell subsets [44]. Plasmid DNA represents an attractive strategy for developing new vaccines against pathogenic fungi. Recently, the ®rst report on a DNA vaccination of a fungal disease has been described [9], in which a plasmid containing the cDNA of antigen 2 of Coccidioides immitis was used. A satisfactory protection against lethal challenge by arthroconidia was obtained. To our knowledge, ours is the ®rst attempt to use genetic vaccination against PCM. The present results stimulate further studies to improve the protective immune response by coimmunization of the gp43 cDNA and cytokine genes such as IL-12, as has been used in other systems [45].

Acknowledgements This work was supported by CNPq, PRONEX and FAPESP (1995/0559-8). We thank R.H. Zaugg of Vical Inc., for supplying the VR-1012 plasmid used to prepare the DNA vaccine.

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