Microbes and Infection 10 (2008) 1251e1258

www.elsevier.com/locate/micinf

Original article

Additive effect of P10 immunization and chemotherapy in anergic

mice challenged intratracheally with virulent yeasts
of Paracoccidioides brasiliensis
Alexandre F. Marques a, Marcelo B. da Silva a, Maria A.P. Juliano b, Julian E. Munh~oz a,
Luiz R. Travassos c, Carlos P. Taborda a,*
a

Institute of Biomedical Sciences, Department of Microbiology, University of S~ao Paulo, S~ao Paulo, SP, Brazil
b
Department of Biophysics, Federal University of S~ao Paulo, S~ao Paulo, SP, Brazil
c
Department Microbiology, Immunology and Parasitology, Federal University of S~ao Paulo, S~ao Paulo, SP, Brazil
Received 13 May 2008; accepted 8 July 2008
Available online 24 July 2008

Abstract
Paracoccidioidomycosis is a systemic granulomatous disease manifested in the acute/subacute or chronic forms. The anergic cases of the
acute/subacute form are most severe, leading to death threatening conditions. Drug treatment is required to control the disease but the response in
anergic patients is generally poor. A 15-mer peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4 helper-1 immune
response in mice of different haplotypes and protects against intratracheal challenge with virulent P. brasiliensis. Presently, P10 immunization
and chemotherapy were associated in an attempt to improve antifungal treatment in Balb/c mice made anergic by adding dexamethasone to the
drinking water. The combined drug/peptide treatment significantly reduced the lung CFUs in infected anergic mice, largely preserved lung
alveolar structure and prevented fungal dissemination to liver and spleen. Results recommend that a P10-based vaccine should be associated to
chemotherapy for improved treatment of paracoccidioidomycosis aiming especially at anergic cases.
! 2008 Elsevier Masson SAS. All rights reserved.
Keywords: P. brasiliensis; Anergy; Dexamethasone; Chemotherapy; Immunization; P10; gp43

1. Introduction
Paracoccidioidomycosis (PCM) is a systemic granulomatous
disease caused by Paracoccidioides brasiliensis, a thermally
dimorphic fungus widespread in Latin America mainly
affecting rural workers. An estimate on the incidence of PCM in
Latin America showed that approximately 10 million people
may be infected and 2% of them may develop the disease [1].
The incidence may increase in recent areas of deforestation and
development with the practice of soil churning, as well as in
immunocompromised individuals [2]. In the 1980e1995 period
* Corresponding author. Instituto de Ciencias Biomedicas II, Departamento
de Microbiologia, Universidade de S~ao Paulo, Av. Prof. Lineu Prestes, 1374,
2" andar, S~ao Paulo, SP 05508-900, Brazil. Tel.: 55 11 3091 7345; fax: 55
11 3091 7354.
E-mail address: taborda@usp.br (C.P. Taborda).
1286-4579/$ - see front matter ! 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.micinf.2008.07.027

there occurred 3181 lethal cases only in Brazil, but this is

probably underestimated, since the disease is not classified as of
mandatory notification by the governmental health authority [3].
The disease can exhibit two forms, acute/subacute and
chronic. The former equally affects both genders and primarily
involves the reticuloendothelial/lymphatic system. The chronic
form affects mainly adult males with predominant pulmonary
and/or mucocutaneous involvement [4]. Activation of an
immune cellular response is the primary effective mechanism of
control of experimental and human PCM and a correlation has
been found between the severity of the disease and impaired
delayed-type hypersensitivity (DTH) response [5].
Antifungal chemotherapy is required to control the disease.
Initial treatment with sulfonamides, amphotericin B, or azoles
may last from 2 to 6 months. Extended periods of treatment
are often necessary, up to 2 or more years [6]. Significant
frequency of relapsing disease is observed in patients

1252

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

submitted to short periods of treatment (reviewed in ref. [7]).

Anergic conditions are death threatening due to poor response
to chemotherapy when the immune system collapses.
The gp43 is the major diagnostic antigen of P. brasiliensis. Antibodies to the glycoprotein can be used in the follow-up of patients
using a variety of serological tests [8,9]. Also, it was demonstrated that
gp43 can elicit delayed hypersensitivity reactions in guinea pigs [10]
and humans [11], implying the presence of T-CD4 reacting epitopes.
The gp43 gene was cloned and sequenced encoding a polypeptide of 416 amino acids (Mr 45,947) with 56e58% similarity
with exo-1,3-b-D-glucanases from Saccharomyces cerevisiae
and Candida albicans [12]. The mature protein has a single high
mannose N-glycosylated site [13]. Investigation of the gp43
gene polymorphism showed that the P10 encoding sequence is
highly conserved with rare mutations in this region [14].
The H-2d-restricted T-cell epitope was mapped to a 15-mer
peptide called P10. An inner core of P10 formed by HTLAIR is
the essential domain of the epitope inducing proliferation of
lymph node cells from mice sensitized to the gp43 or infected
with P. brasiliensis. Lymphoproliferation induced by either P10
or gp43 involved CD4T-helper lymphocytes producing IL-2
and IFN-g. Immunization of mice with both gp43 and P10 in the
presence of complete Freunds adjuvant (CFA) caused a 200fold decrease in CFU from the lung of mice challenged intratracheally with virulent yeasts of P. brasiliensis [15].
Recently we have shown that P10 immunization associated to
chemotherapy improved antifungal treatment and prevented
relapses. In all cases the combined immunochemotherapy
showed an additive protective effect clearly superior to drug and
P10-vaccine administered alone. P10-vaccination could
successfully reverse the relapsing infection observed in sulphamethoxazole/trimethoprim treated mice. Generally, drug
treatment favored a Th-2 response producing IL-4 and IL-10,
whereas immunization with P10 stimulated a pro-inflammatory
Th-1 response with increased IL-12 and IFN-g. In successfully
treated mice both Th-1 and Th-2 cytokines were present. The
combined drug/vaccine treatment significantly reduced the lung
CFUs, largely preserved lung alveolar structure and prevented
fungal dissemination to liver and spleen [16].
In the present work we investigated the effectiveness of P10
in an experimental model that aimed at mimetizing the anergic
state, common in patients with the acute and sub acute forms.
In general, we studied the behavior of immunosuppressed
animals, infected and treated with itraconazole or sulfamethoxazole and trimethoprim, associated or not with the
immunization with P10. This novel treatment was shown to be
quite effective in mice intratracheally infected with this fungus
and with the perspective of potentially reducing the time of
treatment in human patients, prevent relapsing disease and
eventually reverse life threatening anergic cases.

conditions. Procedures involving animals and their care were

conducted in conformity with the local Ethics Committee and
international recommendations.
2.2. Fungal strain
Virulent P. brasiliensis Pb18 yeast cells were maintained by
weekly passage on solid Sabouraud medium at 37 " C and were
used after 7e10 days of growth. Before experimental infection, the cultures were grown in modified McVeigheMorton
medium (MMcM) at 37 " C for 5- to 7-days [17]. The fungal
cells were washed in phosphate-buffered saline (PBS, pH 7.2)
and counted in a hemocytometer. The viability of fungal
suspensions determined by staining with trypan blue (Sigma,
St Louis, MO) was always higher than 90%. The virulence of
isolate Pb18 was checked in each experiment by infecting
intratraqueally Balb/c mice and recovering the yeast cells from
the organs of mice.
2.3. Immunosuppression of mice
To examine the effects of immunosuppression in mice
intratracheally infected with P. brasiliensis, dexamethasone
phosphate (Sigma, St Louis, MO) was added to the drinking
water of infected animals. Assuming an average water intake
of 5 ml per day for 30 days, the daily dexamethasone phosphate dose was calculated as 0.15 mg kg#1.
2.4. Delayed-type hypersensitivity (DTH) reaction
To verify the effect of dexamethasone administration the
DTH reactions against Pb-18 antigen were measured as well
the evolution of the infectious process was evaluated. The left
hind footpad was injected subcutaneously with 25 ml of Fava
Nettos antigen every 3 days [18] and variations in size
(diameters) were measured after 24 and 48 h.
2.5. Peptide synthesis and purification
Peptide synthesis and purification was carried out at the
Department of Biophysics, UNIFESP as described previously
[15]. HPLC analysis showed that the synthetic P10 in the
amidated form was 90% pure.
2.6. Immunization of mice

2. Materials and methods

Anergic Balb/c mice (6- to 8-week old males) were weekly

immunized for 4 weeks with 20 mg of P10 each. The first
immunization was subcutaneous with complete Freunds
adjuvant (CFA) and subsequently peptide immunization was
i.p. with incomplete Freunds adjuvant (IFA). Control mice
were injected with CFA alone.

2.1. Animals

2.7. Intratracheal infection of Balb/c mice

Balb/c mice (6- to 8-week-old males) were bred at University of S~ao Paulo animal facility under specific-pathogen-free

Balb/c mice were inoculated intratracheally with 3 $ 105

yeast cells of virulent P. brasiliensis Pb18, grown on

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

Sabouraud agar and suspended in sterile saline (0.85% NaCl),

per animal. A maximum volume of 50 ml was inoculated per
mouse. Briefly, mice were anesthetized i.p. with 200 mL of
a solution containing 80 mg/kg of ketamine and 10 mg/kg of
xylazine (both from Uni~ao Qumica Farmaceutica, Brazil);
after approximately 10 min, their necks were hyper-extended,
and the trachea was exposed at the level of the thyroid and
injected with 3 $ 105 yeast cells in PBS using a 26-gauge
needle. The incisions were sutured with 5-0 silk.

1253

homogenized in 2 ml of PBS in the presence of protease

inhibitors (Complete Mini; Boehringer Mannheim, Indianapolis, IN). The homogenates were centrifuged, and the
supernatants frozen at #80 " C until tested. The supernatants
were assayed for IL-2, IL-4, IL-10, IL-12, and IFN-g using
ELISA kits (BD PharMingen, San Diego, CA). The detection
limits of such assays were as follows: 3.1 pg/ml for IL-2;
7.8 pg/ml for IL-4; 31.25 pg/ml for IL-10 and IFN-g; and
62.5 pg/ml for IL-12p40, as previously determined by the
manufacturer.

2.8. Fungal burden in organs of infected mice

2.12. Antibody response to gp43
Mice were sacrificed 45 days after i.t. infection and the
fungal burden was measured by colony forming units (CFU).
Sections of the lung, liver and spleen were removed, weighed,
homogenized and then washed three times with PBS. The corresponding pellets were re-suspended and homogenized each in
1 ml of PBS. A 100 ml-sample of this suspension was plated on
solid braineheart infusion (BHI) medium supplemented with
4% fetal calf serum (Gibco, NY, USA), 5% spent P. brasiliensis
isolate-192 culture supernatant, streptomycin/penicillin 10 IU/
ml (Cultilab, Brazil) and cycloheximide 500 mg/ml (Sigma, St
Louis, MO). Petri dishes were incubated at 37 " C for at least
10 days, and colonies were counted (1 colony 1 CFU).

The antibody response to gp43 was determined 45 days

after infection. Microtiter plates sensitized with 500 ng of
purified gp43 were incubated at 37 " C for 1 h with 100 ml of
mouse serum, serially diluted starting at 1:200. The reaction
was quantified with goat anti-mouse Ig for 1 h at 37 " C, followed by donkey anti-goat Ig-biotin conjugate and reaction
with streptavidin-peroxidase for 30 min at 37 " C. After addition of the orthophenylenediamine reagent (500 mg/ml), the
reaction was read at 492 nm. The antibodies, conjugates, and
substrate were from Sigma Chemical Co (St Louis, MO).
2.13. Histopathology

2.9. Chemotherapy of anergic infected mice

The anergic state was obtained 20 days after the administration of dexamethasone. Animals were then infected and
drug treatment started 15 days after i.t. infection. The treatment was held for 30 days during which groups of mice
received doses every 24 h of itraconazole (10 mg/kg), or sulfamethoxazole/trimethoprim (15 mg and 3 mg/kg). All drug
administrations were i.p.
2.10. Groups studied
Eight groups of mice (with 10 animals each) were used.
Three controls were included: sham, a group that was neither
infected nor treated with dexamethasone, antifungals or with
P10; a second control group was infected with Pb 18, received
dexamethasone in the drinking water but was not further
treated with either an antifungal drug or P10; a third control
group included mice infected but not treated with dexamethasone, antifungal drugs or P10. Five anergic groups included:
(a) a group infected and immunized with P10; (b) two groups
of mice infected and treated with either itraconazole or sulphamethoxazole/trimethoprim; and (c) two groups treated with
antifungal drugs and also immunized with P10. All five groups
(anergic) received dexamethasone in the drinking water. The
whole experiment was repeated twice with reproducible
results.
2.11. Cytokine analysis
Mice were sacrificed 45 days after infection and sections of
the lung (right and left alternatively), liver and spleen were

The lungs were excised, fixed in 10% buffered formalin,

and embedded in paraffin for sectioning. The sections were
stained with hematoxylin-eosin or silver nitrate and examined
microscopically at 25$ magnification (Optiphot-2; Nikon,
Tokyo, Japan).
2.14. Statistical analysis
Statistical analysis was done using GraphPad Prism5 software. The results were expressed as means & SD. The nonparametric Tukey test was used. Unpaired Students t-test with
Welchs correction (two-tailed) was used for the comparison
of two groups when the data met the assumptions of the t tests.
p < 0.05 indicated statistical significance. Survival experiments were compared by log rank analysis (SigmaStat; SPSS,
Chigaco, IL).
3. Results
3.1. Determination of the anergic state in Balb/c mice
A group of infected animals were treated with dexamethasone in the drinking water and every 3 days the animals were
submitted to a DTH test to determine the time necessary for
complete anergy. No DTH reaction was observed after 20 days
of treatment with dexamethasone (Fig. 1A). The mortality in
groups of infected mice submitted or not to dexamethasone
treatment was scored up to 100 days after infection. It was
restricted to the group of animals that received dexamethasone
phosphate in the drinking water in the time range of the
experiment (Fig. 1B). Unlike the control group, all animals

1254

Foot pad (mm)

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

3

liver of animals (CFU) was obtained in mice immunized with

P10. The treatment with itraconazole and sulfamethoxazole/
trimethoprim also reduced the fungal burden; a more significant
reduction, however, was obtained when itraconazole or sulfamethoxazole/trimethoprim and P10 immunization were
combined in the treatment of anergic Balb/c mice (Fig. 2).

Anergy
Control

3.3. Lung histopathology from anergic Balb/c mice

vaccinated with P10 only, after intratracheal infection

10

15

20

25

Days of infection

B 100
Anergy
Control

% Survival

80

60

40

Lungs of infected animals submitted to dexamethasone

treatment showed multiple pulmonary foci of epithelioid granulomatous inflammation, with a loose architecture containing
a great number of neutrophils and fungal cells (Fig. 3A). Lungs of
animals submitted to P10 immunization showed few well
demarcated epithelioid granulomas, compact and with few
fungal cells (Fig. 3B). A similar pattern of lung histopathology
was observed in the tissues of mice treated with itraconazole
(Fig. 3C) or sulfamethoxazole/trimethoprim (Fig. 3E). The
association of drugs and immunization with P10 resulted in areas
with very few or undetectable yeast cells (Fig. 3D,F). Clearly, in
this case, P10-immunization was an essential adjuvant therapy
that reduced the infection. Few untreated animals showed, during
the experiment, brain dissemination of fungal elements that was
absent in the P10 immunized mice (data not shown).

20

20

30

40

50

60

70

80

90

3.4. Cytokine detection in the lungs of anergic infected

Balb/c mice immunized with P10 and/or treated with
antifungal drugs

Days of infection
Fig. 1. Delayed-type hypersensitivity and Survival curve of Balb/c mice. (A)
Follow-up of DTH reactions in infected Balb/c mice (B) and infected,
immunosuppressed Balb/c mice (C). Dotted line represents averages of
uninfected Balb/c mice. Error bars denote standard deviations. (B) Survival of
Balb/c mice infected with 3 $ 105 yeast cells of P. brasiliensis Pb18 treated
with dexamethasone (0.15 mg/kg) in the drinking water for 30 days (C);
untreated control (B). The experiments were repeated twice with similar
results.

were killed by day 70. After determining the time necessary

for anergy induction, all subsequent experimental infections
were carried out after 20 days of exposure to dexamethasone
in the drinking water. Infected anergic mice submitted to P10
vaccination and/or antifungal drug treatment were all alive in
the 100-day period of the experiment much like the control
mice untreated with dexamethasone (data not shown).
3.2. Organ CFU from intratracheally infected anergic
Balb/c mice immunized with P10 and/or treated with
antifungal drugs
Our previous work has shown that P10 could be used as
adjuvant in the treatment of established experimental infection
[16]. To explore the effects of P10 immunization in anergic Balb/
c mice, animals immunization started 15 days after infection.
Analysis of organ CFUs was done after 45 days of infection. A
significant reduction of fungi recovered from lung, spleen and

Previous studies have established that P10 was able to activate

a Th-1 immune response with the known pattern of cytokine
production in vitro and in vivo [15,16]. Presently, lung cytokine
levels were measured in anergic mice infected with P. brasiliensis
and treated with itraconazole or sulfamethoxazole/trimethoprim
combined or not with P10 immunization. A sham group was
evaluated for basal levels of cytokines as well as a control group of
infected-only mice untreated with dexamethasone, antifungal
drugs or peptide as shown in Table 1. The comparative cytokine
levels among infected Balb/c mice treated or not with dexamethasone were then evaluated. Results showed that infected mice
treated with dexamethasone had a much higher concentration of
IL-4 and IL-10 than of IL-12 and IFN-g. Treatment of mice with
antifungal drugs led to partial reduction of IL-4 and IL-10 levels
which were then further reduced with the association of P10
immunization and the antifungal drug. P10 immunization as well
as its association with antifungal treatment markedly enhanced
IL-12 and IFN-g levels. Thus, although both Th-1 and Th-2 types
of immune response seemed present in all cases, incorporation of
P10 greatly stimulated a Th-1 response with increased production
of IL-12 and IFN-g, clearly associated with protection.
3.5. Antibody titers against gp43 after 45 days
of infection
Before sacrifice for other determinations, blood was
collected from infected animals and the serum antibodies to

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

1255

Fig. 2. Colony forming units from organs of infected mice. Colony forming units (CFU) from lungs (L), spleen (S) and liver (V) of Balb/c mice infected
intratracheally with 3 $ 105 yeast cells of P. brasiliensis Pb18 (non-anergic) or infected and treated with dexamethasone (anergic). Groups of anergic animals were
also submitted to P10 immunization (P10), itraconazole (Itra) or sulfamethoxazole/trimethoprim (SMT/TMP) treatment, or the combined P10 immunization and
chemotherapy (itraconazole P10 and SMT/TMP P10). Bars represent the CFU means and standard deviations from organs of five to 10 animals in each group.
* significant ( p < 0.05) difference in relation to the group of mice infected and treated only with dexamethasone in drinking water. ** significant ( p < 0.05)
differences between the combined treatment of P10 and antifungal drug, and the individual treatments with each agent.

the gp43 major antigen were titrated. Animals treated with

dexamethasone and infected, showed after 45 days by ELISA
an antibody titer of 1/6400 compared to 1/200 in mice infected
and submitted to immunization with P10. Anergic mice
immunized with P10 and treated with either itraconazole or
sulfamethoxazole/trimethoprim also produced fewer specific
antibodies than those treated only with antifungal drugs.
4. Discussion
The main clinical forms of PCM are: acute/subacute and
chronic. The acute form is more severe and represents 3e5%
of all cases of PCM affecting mainly children and teenagers
but eventually also adults up to 35 years old (reviewed in ref.
[6]).
Clinical evidence as well as experimental studies have
shown that cell-mediated immunity is the chief mechanism of
host defense. Acute/subacute forms are more frequently
associated with anergic immune response whereas the chronic
form is hyperergic. Acute/subacute forms tend to show
necrotizing lesions with abundant fungal cells, impairment of
cell-mediated immunity and high titers of circulating antibodies. In contrast, the chronic form is seldom disseminated,
and is associated with tuberculoid granulomata and few fungal
cells. The cell-mediated immunity is better preserved with low
antibody levels [19,20].
The control of the disease is made by antifungal drugs such
as sulphonamides, amphotericin B and azoles (reviewed in ref.
[7]). Itraconazole is a good option to start the treatment but
sulphamethoxazole/trimetoprim therapy is still being used in
public hospitals in Brazil [6].
The use of peptides as therapeutic vaccines in the treatment
of fungal infections as they have being used in cancer [21]
could be an adjuvant to chemotherapy potentially able to
reduce the toxicity of conventional drugs as well as the time of

treatment, prevent relapsing disease and even help to treat

anergic cases [22,23].
The immunoprotective properties of P10 were well determined in a murine model of PCM. Injection of P10 emulsified
in complete Freunds adjuvant (CFA) protected mice against
intratracheal challenge with virulent P. brasiliensis isolate.
Disease progression was followed by counting the CFUs in
tissues and by histopathology. The protective effect of P10 is
undoubtedly related to the induction of an IFN-g-dependent
Th-1 immune response [15]. A P10-derived multiple-antigenpeptide construction (MAP) administered without CFA
conferred equal protection in i.t. infected mice [24]. We have
shown then that an additive protective effect was achieved by
the combination of drug treatment and P10 immunization [16].
Aiming at defining the role of P10 as an adjuvant immune
treatment in drug treated, infected animals, we presently
extended this study to determine the capacity of P10 to reverse
the anergic state of Balb/c mice induced by dexamethasone.
The recognized anergic state was observed after 20 days of
treatment with dexamethasone in the drinking water a condition that caused death of all infected mice after 70 days of
infection. The use of dexamethasone to induce the anergic
state in mice has been described previously [25].
Anergic animals i.t. infected with virulent P. brasiliensis
showed high CFU in the lungs and also in the liver, spleen and
brain after 45 days of infection. Cytokine determination
showed increased IL-4 and IL-10 and low levels of IFN-g and
IL-12, a pattern that was reversed by P10 immunization and
less efficiently by antifungal drugs. Combination of P10 and
antifungal drugs promoted the best Th-1 type immune
conversion. Anti-gp43 IgG titers were higher in the dexamethasone and antifungal treated groups than in the P10immunized ones. Immunization of anergic Balb/c mice with
P10 led to significant reduction of CFU in the lung, spleen and
liver. The protective properties of IFN-g in the murine model

1256

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

Fig. 3. Histopathology of lungs from intratracheally infected anergic Balb/c mice submitted to immunization with P10 and/or chemotherapy. Additive protective
effect of P10 immunization and antifungal drug treatment is shown after 45 days of infection. (A) Lung section from infected mouse treated only with dexamethasone; (B) same as (A) but immunized with P10, showing a single granuloma; (C) lung section from anergic mouse treated with intraconazole; (D) same from
anergic mouse immunized with P10 and treated with itraconazole; (E) lung section form anergic mouse treated with sulfamethoxazole/trimetoprim (SMT/TMT)
and (F) same from anergic mouse immunized with P10 and treated with (SMT/TMT). Hematoxylin eosin staining; magnification, $25.

have been established previously. Survival analysis of mice

deficient in IFN-g or IFN-g receptor (but not IFN-a-R or IFNb-R), and IRF-1 showed 100% mortality 3e4 weeks after
intratracheal challenge with a virulent isolate. Consistent with
the need of IFN-g for effective immunization with P10, nonimmunized infected IFN-g-R knock-out mice displayed the
same mortality rate as of P10-immunized KO mice [9].

Histopathology of infected organs confirmed the protective

effect of P10, either by significantly reducing the number of
granulomas or, when associated with antifungal drugs, leading
to fungal elimination but not sterility because of the short time
of the experimental design [26].
The use of P10 as an adjuvant to antifungal treatment is a
promising tool in the treatment of experimental

1257

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

Table 1
Lung cytokine levels after 45 days of infection in anergic Balb/c mice infected with 3 $ 105 yeast cells of P. brasiliensis Pb18
Cytokine Cytokine levels (ng/ml of tissue)
Shama
IL-2
IL-4
IL-10
IL-12
IFN-g

Infected onlyb Infected dexamethasonec P10d

1.71 & 0.7

2.51 & 0.56
0.2 & 0.06 3.37 & 0.69
4.39 & 1.87 12.52 & 1.76
0.58 & 0.23 2.12 & 0.50
0.38 & 0.26 0.31 & 0.11

1.98 & 0.94

10.9 & 1.86
20.16 & 5.24
1.52 & 0.25
1.1 & 0.7

8.09 & 0.32

1.14 & 0.46
8.16 & 2.99
10.48 & 1.53
4.5 & 0.88

Itraconazolee Itraconazole and P10f SMT/TMPg

3.16 & 1.0
4.86 & 0.38
8.66 & 2.52
2.81 & 0.43
1.18 & 0.30

8.07 & 1.98*

0.68 & 0.19*
3.64 & 2.04*
11.6 & 1.38*
7.39 & 1.34*

SMT/TMP and P10h

3.4 & 1.35 6.51 & 0.56*

5.56 & 0.61 0.62 & 0.12*
13.28 & 2.64 6.53 & 2.16*
2.2 & 0.48 10.05 & 0.63*
0.41 & 0.22 6.54 & 0.77*

*Significant differences ( p < 0.05) relative to anergic mice submitted to chemotherapy only. Values are means for 10 animals per group with standard deviations.
The whole experiment was repeated twice with reproducible results.
a
Uninfected, non-immunized and untreated animals (n 10).
b
Infected only, untreated with dexamethasone, antifungal drugs or P10.
c
Infected only, but treated with dexamethasone.
d
Treated with dexamethasone (anergic), infected and immunized with P10.
e
Anergic, treated with itraconazole.
f
Anergic, treated with itraconazole and P10 immunization.
g
Anergic, treated with sulfamethoxazole/trimetoprim (SMT/TPM).
h
Anergic, with SMT/TPM and P10 immunization.

paracoccidioidomycosis with a role also in the prevention of

relapses. The combined treatment could be important even in
cases of clinical resistance to azoles and sulfamethoxazole/
trimethoprim [7,16]. Presently, we report on the additive
protective effect of itraconazole or sulfamethoxazole/trimethoprim and P10 immunization in anergic animals. Anergic
Balb/c mice treated only with antifungal drugs still showed
high levels of IL-4 and IL-10 possibly due to increased fungal
destruction and high B-cell stimulation. The combined treatment with a P10-vaccine clearly stimulated a protective Th-1
response rich in IL-12 and IFN-g. A predominant Th-1
immune response simultaneous with a persisting but less
intense Th-2 response seems to represent the adequate balance
leading to protection of infected animals. Most probably Tdependent antibodies are also implicated in the immunoprotection but with the exception of anti-gp43 antibodies which
may include protective and non-protective types [27] this was
not further explored presently.
Several features have been examined for validation of P10 as
a candidate vaccine antigen including: (a) the presentation of
P10 by different mouse haplotypes [15]; (b) its conservation in
nature determined by examining gp43 molecules from different
isolates [28]; (c) its immunogenicity in formulations that do not
require complete Freunds adjuvant [24,29]; (d) its presentation
by human HLA-DR molecules and that of other promiscuous
peptides derived from the gp43 [30]; and (e) additive effect when
administered with antifungal drugs [16] and finally, its role in the
immunoprotection of anergic mice infected with P. brasiliensis
and submitted to chemotherapy which was the focus of the
present work. The present data strongly suggest that a P10-based
vaccine may be used to enhance antifungal protection by
chemotherapy even in cases of anergy.
Acknowledgements
CPT and LRT were supported by Fundac~ao de Amparo a`
Pesquisa do Estado de S~ao Paulo (Fapesp) grants 07/07588-2

and 06/50634-2, CNPq grant 470636/2007-6. CPT and LRT

are research fellows of the CNPq.

References
[1] J.G. McEwen, A.M. Garcia, B.L. Ortiz, S. Botero, A. Restrepo, In search
of the natural habitat of Paracoccidioides brasiliensis, Arch. Med. Res.
26 (1995) 305e306.
[2] B. Wanke, A.T. Londero, Epidemiology and paracoccidioidomycosis
infection, in: M. Franco, C.S. Lacaz, A. Restrepo, G. del Negro (Eds.),
Paracoccidioidomycosis, CRC press, Boca Raton, Florida, 1994, pp.
109e117.
[3] Z.F. Coutinho, D. Silva, M. Lazera, V. Petri, R.M. Oliveira, P.C. Sabroza,
B. Wanke, Paracoccidioidomycosis mortality in Brazil (1980e1995),
Cad. Saude Publica 18 (2002) 1441e1454.
[4] M. Franco, Host-parasite relationships in paracoccidioidomycosis, J.
Med. Vet. Mycol. 25 (1987) 5e18.
[5] C.C. Musatti, M.T.S. Peracoli, A.M.V.C. Soares, M.T. Rezkallah-Iwasso,
Cell-mediated immunity in patients with paracoccidiodomycosis, in:
M. Franco, C.S. Lacaz, A. Restrepo, G. del Negro (Eds.), Paracoccidioidomycosis, CRC Press, Boca Raton, Florida, 1994, pp.
175e186.
[6] M.A. Shikanai-Yasuda, Q. Telles Filho Fde, R.P. Mendes, A.L. Colombo,
M.L. Moretti, Guidelines in paracoccidioidomycosis, Rev. Soc. Bras.
Med. Trop. 39 (2006) 297e310.
[7] R.C. Hahn, Y.T. Morato Conceicao, N.L. Santos, J.F. Ferreira,
J.S. Hamdan, Disseminated paracoccidioidomycosis: correlation between
clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole, Mycoses 46 (2003) 342e347.
[8] C.F. Albuquerque, S.H. da Silva, Z.P. Camargo, Improvement of the
specificity of an enzyme-linked immunosorbent assay for diagnosis of
paracoccidioidomycosis, J. Clin. Microbiol. 43 (2005) 1944e1946.
[9] L.R. Travassos, C.P. Taborda, L.K. Iwai, E. Cunha Neto, R. Puccia, The
gp43 from Paracoccidioides brasiliensis: a major diagnostic antigen and
vaccine candidate, in: J.E. Domer, G.S. Kobayashi (Eds.), The Mycota
XII, Human Fungal Pathogens, Springer-Verlag, Berlin-Heidelberg,
2004, pp. 279e296.
[10] E.G. Rodrigues, L.R. Travassos, Nature of the reactive epitopes in Paracoccidioides brasiliensis polysaccharide antigen, J. Med. Vet. Mycol. 32
(1994) 77e81.
[11] E.C. Saraiva, A. Altemani, M.F. Franco, C.S. Unterkircher,
Z.P. Camargo, Paracoccidioides brasiliensis-gp43 used as paracoccidioidin, J. Med. Vet. Mycol. 34 (1996) 155e161.

1258

A.F. Marques et al. / Microbes and Infection 10 (2008) 1251e1258

[12] P.S. Cisalpino, R. Puccia, L.M. Yamauchi, M.I. Cano, J.F. da Silveira,
L.R. Travassos, Cloning, characterization, and epitope expression of the
major diagnostic antigen of Paracoccidioides brasiliensis, J. Biol. Chem.
271 (1996) 4553e4560.
[13] I.C. Almeida, D.C. Neville, A. Mehlert, A. Treumann, M.A. Ferguson,
J.O. Previato, L.R. Travassos, Structure of the N-linked oligosaccharide
of the main diagnostic antigen of the pathogenic fungus Paracoccidioides
brasiliensis, Glycobiology 6 (1996) 507e515.
[14] F.V. Morais, T.F. Barros, M.K. Fukada, P.S. Cisalpino, R. Puccia, Polymorphism in the gene coding for the immunodominant antigen gp43
from the pathogenic fungus Paracoccidioides brasiliensis, J. Clin.
Microbiol. 38 (2000) 3960e3966.
[15] C.P. Taborda, M.A. Juliano, R. Puccia, M. Franco, L.R. Travassos,
Mapping of the T-cell epitope in the major 43-kilodalton glycoprotein of
Paracoccidioides brasiliensis which induces a Th-1 response protective
against fungal infection in BALB/c mice, Infect. Immun. 66 (1998)
786e793.
[16] A.F. Marques, M.B. da Silva, M.A. Juliano, L.R. Travassos,
C.P. Taborda, Peptide immunization as an adjuvant to chemotherapy in
mice challenged intratracheally with virulent yeast cells of Paracoccidioides brasiliensis, Antimicrob. Agents Chemother. 50 (2006)
2814e2819.
[17] A. Restrepo, M.D. Arango, In vitro susceptibility testing of Paracoccidioides brasiliensis to sulfonamides, Antimicrob. Agents Chemother. 18 (1980) 190e194.
[18] C. Fava Netto, V.S. Vegas, J.M. Sciannamea, E.D.B. Guarnieri, Antgeno
polissacardico do Paracoccidioides brasiliensis. Estudo do tempo de
cultivo necessario ao preparo do antgeno, Inst. Med. Trop. S~ao Paulo 11
(1969) 177e181.
[19] G. Del Negro, C.S. Lacaz, V.A. Zamith, A.M. Siqueira, General
clinical aspects: polar forms of paracoccidioidomycosis, the disease in
childhood, in: M. Franco, C.S. Lacaz, A. Restrepo, G. del Negro
(Eds.), Paracoccidioidomycosis, CRC Press, Boca Raton, Florida,
1994, pp. 225e232.
[20] M. Franco, M.R. Montenegro, R.P. Mendes, S.A. Marques, N.L. Dillon,
N.G. Mota, Paracoccidioidomycosis: a recently proposed classification of
its clinical forms, Rev. Soc. Bras. Med. Trop. 20 (1987) 129e132.
[21] C.L. Slingluff Jr., G.R. Petroni, G.V. Yamshchikov, S. Hibbitts,
W.W. Grosh, K.A. Chianese-Bullock, E.A. Bissonette, D.L. Barnd,
D.H. Deacon, J.W. Patterson, J. Parekh, P.Y. Neese, E.M. Woodson,
C.J. Wiernasz, P. Merrill, Immunologic and clinical outcomes of

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

vaccination with a multiepitope melanoma peptide vaccine plus low-dose

interleukin-2 administered either concurrently or on a delayed schedule,
J. Clin. Oncol. 22 (2004) 4474e4485.
L.R. Travassos, A. Casadevall, C.P. Taborda, Immunomodulation and
immunoprotection in fungal infections: humoral and cellular immune
responses, in: G. San-Blas, R.A. Calderone (Eds.), Pathogenic Fungi:
Host Interactions and Emerging Strategies for Control, Caister Academic
Press, Norfolk, England, 2004, pp. 241e283.
L.R. Travassos, G. Goldman, C.P. Taborda, R. Puccia, Insights in Paracoccidioides brasiliensis pathogenicity, in: K. Kavanagh (Ed.), New
Insights in Medical Mycology, Springer, Dordrecht, The Netherlands,
2007, pp. 241e265.
C.P. Taborda, C.R. Nakaie, E.M. Cilli, E.G. Rodrigues, L.S. Silva,
M.F. Franco, L.R. Travassos, Synthesis and immunological activity of
a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis, Scan. J. Immunol. 59
(2004) 58e65.
L.H. Elliott, A.K. Levay, Costimulation with dexamethasone and prostaglandin E2: a novel paradigm for the induction of T-cell anergy, Cell
Immunol. 180 (1997) 124e131.
L.R. Travassos, E.G. Rodrigues, L.K. Iwai, C.P. Taborda, Attempts at
a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy, Mycopathologia 165 (2008) 341e352.
R. Buissa-Filho, R. Puccia, A.F. Marques, F.A. Pinto, J.E. Mu~noz,
J.D. Nosanchuk, L.R. Travassos, C.P. Taborda, The monoclonal antibody
against the major diagnostic antigen of Paracoccidioides brasiliensis
mediates immune protection in infected BALB/c mice challenged
intratracheally with the fungus, Infect. Immun. 76 (2008) 3321e3328.
K.C. Carvalho, L. Ganiko, W.L. Batista, F.V. Morais, E.R. Marques,
G.H. Goldman, M.F. Franco, R. Puccia, Virulence of Paracoccidioides
brasiliensis and gp43 expression in isolates bearing known PbGP43
genotype, Microbes Infect 7 (2005) 55e65.
A.R. Pinto, R. Puccia, S.N. Diniz, M.F. Franco, L.R. Travassos, DNAbased vaccination against murine paracoccidioidomycosis using the gp43
gene from Paracoccidioides brasiliensis, Vaccine 18 (2000) 3050e3058.
L.K. Iwai, M. Yoshida, A. Sadahiro, W.R. da Silva, M.L. Marin,
A.C. Goldberg, M.A. Juliano, L. Juliano, M.A. Shikanai-Yasuda,
J. Kalil, E. Cunha-Neto, L.R. Travassos, T-cell recognition of Paracoccidioides brasiliensis gp43-derived peptides in patients with paracoccidioidomycosis and healthy individuals, Clin. Vaccine Immunol. 14
(2007) 474e476.