Mycopathologia (2014) 178:177188

DOI 10.1007/s11046-014-9801-1

Immunization with P10 Peptide Increases Specific Immunity

and Protects Immunosuppressed BALB/c Mice Infected
with Virulent Yeasts of Paracoccidioides brasiliensis
Julian E. Munoz Vinicius D. Luft Juliana Amorim
Adriana Magalhaes Luciana Thomaz Joshua D. Nosanchuk
Luiz R. Travassos Carlos P. Taborda

Received: 24 September 2013 / Accepted: 7 August 2014 / Published online: 19 August 2014
Springer Science+Business Media Dordrecht 2014

Abstract Paracoccidioidomycosis is a systemic

granulomatous disease caused by Paracoccidioides
spp. A peptide from the major diagnostic antigen
gp43, named P10, induces a T-CD4? helper-1
immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide
alone or combined with antifungal drugs in mice
immunosuppressed and infected with virulent isolate
of P. brasiliensis. In the present work, our data suggest
that P10 immunization leads to an effective cellular
immune response associated with an enhanced T cell
proliferative response. P10-stimulated splenocytes

J. E. Munoz
V. D. Luft
J. Amorim
A. Magalhaes

L. Thomaz
C. P. Taborda (&)
Department of Microbiology, Institute of Biomedical
Sciences, University of Sao Paulo, Av. Prof. Lineu
Prestes, 1374, Sao Paulo, SP 05008-900, Brazil
e-mail: taborda@usp.br
J. D. Nosanchuk
Departments of Medicine, and Microbiology and
Immunology, Albert Einstein College of Medicine,
Bronx, NY, USA
L. R. Travassos
Department of Microbiology, Immunology and
Parasitology, Federal University of Sao Paulo, Sao Paulo,
SP, Brazil
C. P. Taborda
Laboratory of Medical Mycology-LIM53/IMTSP,
University of Sao Paulo, Sao Paulo, SP, Brazil

increased nitric oxide (NO) production and induced

high levels of IFN-c, IL-1b and IL-12. Furthermore,
significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was
followed by minimal fibrosis in response to infection.
Combined with antifungal drugs, P10 immunization
most significantly improved survival of anergic
infected mice. Administration of either itraconazole
or sulfamethoxazole/trimethoprim together with P10
immunization resulted in 100 % survival up to
200 days post-infection, whereas untreated mice died
within 80 days. Hence, our data show that P10
immunization promotes a strong specific immune
response even in immunocompromised hosts and thus
P10 treatment represents a powerful adjuvant therapy
to chemotherapy.
Keywords P. brasiliensis
Anergy
Chemotherapy

P10 immunization

Introduction
The thermally dimorphic fungus Paracoccidioides
brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM). PCM is the most frequent systemic
mycosis in Latin America, with the highest incidence
of diagnosis in Brazil, Argentina, Colombia and
Venezuela [1]. The main route of acquisition is the
inhalation of fungal particles, which usually leads to

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an asymptomatic infection [2]. There are two main

clinical forms of PCM, acute/subacute and chronic.
The acute/subacute form is characterized by a rapid
course (weeks to months), impaired cellular immunity, the absence of delayed-type hypersensitivity
reactions and high mortality rate. The chronic form
affects mainly adult males of 3050 years old with
predominant pulmonary and/or mucocutaneous
involvement [3].
An effective cellular immune response is essential
for the control of experimental and human PCM [4].
Several studies have shown that high levels of specific
antibodies and polyclonal activation of B cells are
associated with the severe form of the disease, whereas
inflammatory cytokines, such as IFN-c, IL-12 and
TNF-a, have an important protective role in the host
resistance [5, 6]. Notably, IFN-c activates macrophages and augments their fungicidal activity against
P. brasiliensis [7].
The 43 kDa glycoprotein (gp43) of P. brasiliensis
is the main diagnostic antigen of PCM as it is
recognized by virtually 100 % of patients sera [8].
The gp43 binds laminin, a protein component of the
extracellular matrix of mammalian tissues, thus facilitating fungal invasion and the subsequent destruction
of tissues [9]. It carries an immunodominant epitope
that induces a predominant IFN-c-mediated Th-1
response [10]. It is primarily responsible for delayedtype hypersensitivity (DTH) reactions in infected
animals [11]. The 15-amino acid peptide of gp43
(QTLIAIHTLAIRYAN), designated as P10, contains
the MHC-II restricted CD4? T cell-specific epitope
which elicits the cellular immune response in BALB/c
mice and other mouse haplotypes [10]. Most importantly, most of the human DR antigens promiscuously
present P10 and neighbor peptides [12].
Treatment of PCM requires intensive and prolonged antifungal chemotherapy. Treatment with
sulfonamides (either sulfamethoxazole or sulfadiazine) combined with trimethoprim, amphotericin B or
an azole is typically administered for 26 months,
although extended periods of treatment (years) are
often necessary depending on the drug employed and
the disease severity [13]. Unfortunately, relapses are
particularly common in patients treated for short
periods [14].
The host immune status can increase the severity of
PCM cases. For example, severe cases of PCM
involving immunosuppressed patients with cancer or

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Mycopathologia (2014) 178:177188

after renal transplantation have been described [15],

presumably due to reactivation of a latent lesion while
administering cytotoxic drugs. PCM occurs in patients
with HIV infection, but at lower incidence compared
to Cryptococcus and Histoplasma mycoses [16].
Notably, PCM patients with HIV infection frequently
develop a rapidly progressive, often multi-focal disease with frequent relapses after initial treatment [17].
HIV patients coinfected with P. brasiliensis have less
immunoreactivity to antigens from this fungus [18].
Corticosteroids such as dexamethasone (Dex) can
be used to treat many different diseases including
cancer. Dex effectively reduces airway inflammation
through multiple mechanisms, including the modulation of the synthesis of inflammatory cytokines [19].
Although P. brasiliensis is not an opportunistic
fungus, cases of infection in patients undergoing
treatment with immunosuppressive drugs have been
reported. Our group has previously shown the additive
protective effect of P10 immunization associated with
antifungal drugs in anergic PCM [20]. Here, we extend
these results to show the survival curve of treated
animals, the cellular immune response and preservation of the lung tissue in P10 vaccination. A
therapeutic peptide vaccine combined with drug
treatment should reduce the time of treatment and in
perspective, avoid disease relapse and drug side
effects.

Materials and Methods

Animals
BALB/c mice, 68-week-old males, five per group,
were housed in polypropylene cages under specific
pathogen-free conditions, and all materials were
sterilized prior to use. Animals used in this study
were bred at Institute of Biomedical Sciences (ICB) of
University of Sao Paulo (USP) animal facility. All
experiments involving animals were conducted and
approved by the Ethics Committee of ICBUSP and
conducted in accordance with international
recommendations.
Immunosuppression of Mice
Dex phosphate (Sigma, St Louis, MO) was used to
induce immune suppression. The corticoid was added

Mycopathologia (2014) 178:177188

179

to the drinking water of animals 20 days before

infection and remained until the day of sacrifice
60 days post-infection. Assuming an average water
intake of 5 ml per day for 30 days, the daily Dex
phosphate dose was calculated as 0.15 mg/kg [20].
Control animals (non-Dex) did not receive Dex
phosphate.

immunization was subcutaneous with P10 in complete

Freunds adjuvant (CFA) followed by i.p. immunization of the peptide in incomplete Freunds adjuvant
(IFA). Control mice were injected with CFA and IFA
alone without the peptide.

Fungal Strain

Antifungal drug treatment started 30 days after i.t.

infection. Thereafter, mice received doses of 10 mg/
kg itraconazole (ITC) (Janssen Pharmaceutica, NV),
or 15 mg sulfamethoxazole plus 3 mg trimethoprim/
kg (SMT/TMP) (Bac-sulfitrin, Ducto, Brazil), every
24 h for 30 days (until day 60, post-infection). All
drug administrations were i.p.

Virulent P. brasiliensis Pb18 yeast cells were used to

infect the animals. The fungal strain was maintained
by weekly passage on solid Sabouraud medium at
37 C. After 710 days of growth, yeast cells were
cultivated in modified McVeigh-Morton medium at
37 C for 57 days [21]. The fungal cells were then
collected, washed in phosphate-buffered saline (PBS
pH 7.2) and counted in a hemocytometer. The viability
of fungal suspensions was determined by staining with
Trypan blue (Sigma, St. Louis, MO) and was always
higher than 90 %. The virulence of the Pb18 strain was
checked in each experiment by intratracheal infection
of BALB/c mice, and recovering the yeast cells from
infected organs.
Intratracheal Infection
BALB/c mice were inoculated i.t. with 3 9 105 yeast
cells/animal of P. brasiliensis Pb18 in sterile saline
(0.85 % NaCl). A maximum volume of 50 ll was
inoculated per mouse. Briefly, the mice were anesthetized i.p. with 200 ll of a solution containing 80 mg/
kg ketamine and 10 mg/kg of xylazine (both from
Uniao Qumica Farmaceutica, Brazil). After approximately 5 min, their tracheas were exposed at the level
of the thyroid and injected with 3 9 105 yeast cells.
Peptide Synthesis and Purification
P10 peptide with amidated C-terminal, used in this
study, was purchased from Peptide 2.0 (Chantilly, VA).
HPLC and MS analyses performed by the manufacturer
showed that the synthetic P10 was 98 % pure.
Immunization of Mice
Anergic BALB/c mice (68 week-old males) were
immunized with 20 lg of P10 once a week for
4 weeks and 30 days after i.t. infection. The first

Treatment with Antifungal Drugs

Groups Studied
Ten groups of mice (n = 5 animals each) were used.
Four controls were included: sham non-Dex, untreated
mice; infected non-Dex, mice infected with Pb 18, but
untreated with steroids; sham, uninfected mice immunosuppressed with Dex phosphate; infected, immunosuppressed mice infected with Pb18 strain; CFA/IFA,
immunosuppressed mice, infected and treated with
CFA/IFA; P10, immunosuppressed and infected mice
immunized with P10 peptide; (ITC), immunosuppressed and infected mice treated with itraconazole;
(SMT/TMP), immunosuppressed and infected mice
treated
with
sulfamethoxazole/trimethoprim;
ITZ ? P10, immunosuppressed and infected mice,
immunized with P10 and treated with itraconazole;
SMT/TMP ? P10, immunosuppressed and infected
mice, immunized with P10 and treated with SMT/TMP.
Cell Proliferation Assay
Spleen cells were collected from mice according to the
different groups to assess P10-stimulated cellular
proliferation. Spleen cells were collected, dispersed
manually and washed in RPMI 1640 (Cultilab, Brazil)
supplemented with 20 mM NaHCO3, 10 mM HEPES,
100 U/ml of penicillin, 100 lg/ml of streptomycin,
2 mM L-glutamine, 50 lM b-mercaptoethanol, 5 mM
sodium pyruvate, 100 mM non-essential amino acids
(Sigma Chemical Co., St. Louis, MO) and 10 % fetal
bovine serum (FBS). Cells were washed twice in FBSfree RPMI, counted, added to 96-well plates at
4 9 105 cells/well and final volume of 200 ll. The

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splenocytes were then stimulated with P10 peptide for

144 h at 37 C in a humidified 5 % CO2 incubator. As
positive control, splenocytes from mice of the sham
group were stimulated with ConA (4 lg/ml, Sigma
Chemical Co., St. Louis, MO). Nave splenocytes
from the sham group were used as negative control. To
determine cell viability/proliferation, MTT (1 mg/ml,
Thiazolyl Blue Tetrazolium BromideSigma, St
Louis) was added to each well during the final 4 h of
culture [22]. The reaction was terminated with 100 ll/
well of isopropanolHCl 0.04 N, and plates were read
in an ELISA reader (Titertek Multiskan EIA reader) at
590 nm wavelength. Data were expressed as means
and standard deviations (SD) of triplicate cultures.
Cytokine Analysis
Cytokines were determined in the supernatants of
splenocyte cultures obtained in the cell proliferation
assay. Interleukin-4 (IL-4), interleukin-12 (IL-12) and
interferon-gamma (IFN-c) were measured using
ELISA kits (BD Biosciences, San Diego, CA), and
interleukin-1b (IL-1b) was measured using an ELISA
kit from eBiosciences, Inc. (San Diego, CA). The
cytokines IFN-c, IL-12, IL-4 and IL-10 from lung
homogenate were also determined using ELISA kits
(BD Biosciences, San Diego, CA). The detection
limits were 7.8 pg/ml for IL-4, 31.25 pg/ml for IFN-c,
62.5 pg/ml for IL-12p40 and 8 pg/ml for IL-1b, as
previously determined by the manufacturer.
Production of Nitric Oxide
Supernatants of the splenocyte cultures obtained in the
cell proliferation assay were used to measure the
production of nitric oxide (NO), in a chemiluminescence analyzer (NOATM280, Sievers Inc., USA). A
calibration curve was set using sodium nitrate standards. Using the NOATM280 analyzer, nitrate was
reduced to NO with vanadium (III) at 90 C and the
NO formed was detected by gaseous phase chemiluminescence after reaction with ozone.
Immunohistochemical Analysis
Lung tissue samples from infected mice were submerged
in liquid nitrogen for 1 min and then stored at -80 C
until analysis. The frozen sections were cut in a cryostat
(Leica CM1850), and sections of 5 micrometers were

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Mycopathologia (2014) 178:177188

applied to poly-L-lysine coated microscope slides (Star

Frost) and fixed with acetone for immunohistochemistry.
After washing with buffer, endogenous peroxidase was
blocked with a 3 % solution of hydrogen peroxide (30 %)
for 5 min. Non-specific protein binding was blocked with
Normal Serum (Vector Laboratories Vectastain ABC
Kit) and BSA 2 % (Bovine Serum Albumin, pH 7.4;
Sigma Chemical Co., St. Louis, MO) was used to block
endogenous biotin. Slides were separately incubated for
an hour with 1:50 (BSA 1 %/Tween 20) rat polyclonal
antibody anti-mouse CD11b, Ly-6G/Ly-6C and L3T4
(BD PharmigenTM San Diego, CA). Biotinylated goat
anti-rat IgG (1:500) (Vector Laboratories, Burlingame,
CA, USA) was used to bind the rat polyclonal antibodies,
applied for 1 h at room temperature, followed by the
addition of streptavidin-peroxidase (1:50) (Vector Laboratories, Burlingame, CA, USA) for 1 h at room
temperature. Chromogen 3,3-diaminobenzidine tetrahydrocloride (DAB; Sigma-Aldrich, St. Louis, MO,
USA) was used to localize peroxidase in tissue sections.
Finally, the slides were counterstained with Mayers
hematoxylin and examined using a light microscope
(Nikon Eclipse E200, Japan).
Fibrosis Evaluation
Lung tissues from infected mice were fixed in 10 %
buffered formalin and then embedded in paraffin for
sectioning. Tissue sections were stained with Gomoris
silver reticulinstain to assess the changes occurring in
the organization of reticulin fibers (collagen III), and
Massons trichromestain to identify collagen I type
fibers. Slides were examined by light microscopy.
Survival Study
Six groups of mice (n = 6 animals each) pre-treated
with Dex were intratracheally infected as described
below: (1) The control group was infected and then
injected only with PBS daily; (2) the second group was
infected and immunized with P10, the first immunization via s.c., with CFA, and subsequently via i.p.,
with IFA; (3) this group was infected and treated with
ITZ; (4) group infected and treated with SMT/TMP;
(5) group infected, immunized with P10 and also
treated with ITZ; and (6) group infected, immunized
with P10 and also treated with SMT/TMP. Deaths
were scored daily for 200 days, and the results were
statistically analyzed.

Mycopathologia (2014) 178:177188

181

Statistical Analysis

Results
Splenocytes from Immunosuppressed Mice
Immunized with P10 Peptide Undergo
Lymphoproliferation When Stimulated with P10
in Vitro
Splenocytes from infected mice, with or without prior
immunosuppression, were exposed in vitro to P10
(Fig. 1). Dex-immunosuppressed animals infected i.t.
for 60 days and with or without treatment with CFA/
IFA or antifungal drugs did not significantly impact
splenocyte proliferation. In contrast, the splenocytes
from immunosuppressed mice that had been immunized with P10 peptide with or without antifungal drug
treatments showed significant proliferation when
stimulated in vitro with P10 when compared with
immunosuppressed and infected animals. These
results indicate that P10 alone strongly stimulated
splenocytes even in immunosuppressed mice after
60 days of infection. As a control, we used splenocytes
stimulated with ConA or infected non-immunosuppressed mice. In both positive control groups, we
observed that splenocyte proliferation was similar to
that achieved in immunosuppressed and P10-stimulated splenocytes, which corresponds to our prior
findings [10].

P10 Induced Proinflammatory Cytokine

Production in Vitro and in Vivo
Previous studies showed that Dex treatment directly
inhibited cytokine production by T cells in mice

**

non-Dex
Dex

D.O. 590

0.4
0.3
0.2
0.1

I
SM TZ
T/
TM
P

P1
P1 0
0+
0+
I
SM TZ
T/
TM
P
P1

on

-s

tim
ul
at
ed
C
on
A
In
fe
ct
ed
In
fe
ct
ed
IF
A/
C
FA

0.0

Statistics was performed using GraphPad Prism5

software (San Diego, CA). The results were expressed
as means and SD. The nonparametric Tukeys honestly significant difference test was employed. p values
of B0.05 indicated statistical significance. In the case
of the survival curve, the Log-rank (MantelCox) test
with p values of B0.0001 was used to indicate
statistical significance. 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.

0.5

Fig. 1 P10 immunization of splenocytes from Dex-immunosuppressed mice significantly increased cellular proliferation.
Splenocytes are isolated from experimental groups after 60 days
i.t. infection with 3 9 105 yeast of P. brasiliensis. Splenocytes
from uninfected mice are incubated in RPMI alone (negative
control) or with ConA (positive control, 4 lg/ml). A second
positive control was the measurement of the proliferation of
splenocytes from infected, untreated mice. The proliferation
rates of splenocytes from immunosuppressed mice with or
without P10 peptide stimulation with or without antifungal
drugs are also assessed. ** p \ 0.01 relative to the control group

infected with P. brasiliensis [23]. Here, we show that

supernatants of splenocyte cultures obtained in the cell
proliferation assay of Dex-treated mice infected with
Pb18 and immunized with P10 produced increased
amounts of IL-12, IFN-c and IL-1b compared to cell
cultures of immunosuppressed mice infected that were
not immunized (control group) (Fig. 2). Notably,
infected, immunosuppressed mice treated with IFA/
CFA also showed an increase in IFN-c similar to that
in P10 immunization. Dex-treated mice, infected with
Pb18 and immunized with P10, showed similar levels
of IL-4 compared to controls (Fig. 2).
Consistent with our prior results [20], although
different times of infection were examined, Table 1
shows that mice treated with Dex that were subsequently infected and immunized with P10 peptide
produced significantly increased amounts of IL-12 and
IFN-c while concomitantly having significant reductions in IL-4 (except SMT/TMP ? P10) and IL-10
when compared with immunosuppressed and infected
mice that were not immunized with P10 (controls).
Results from animals that only received IFA/CFA
adjuvant were similar to that of controls animals
(Table 1). Theses results are compatible with proliferation results shown in Fig. 1.

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Mycopathologia (2014) 178:177188

Fig. 2 Cytokines from

splenocyte cultures of
immunosuppressed BALB/c
mice after 60 days i.t.
infection with 3 9 105 yeast
of P. brasiliensis. (nonDex): Control animals,
untreated with
dexamethasone phosphate.
(Dex): animals treated with
dexamethasone phosphate.
Asterisks indicate
statistically significant
differences between
infected mice immunized
with P10 and unimmunized,
infected animals (*,
p B 0.05; **, p B 0.01)

Nitric Oxide Production

Although Dex treatment inhibited NO production in
macrophages [24], significant concentrations of NO
were detected in splenocyte cultures from all groups of
mice immunized with P10 as compared to controls
(Fig. 3). Treatment with either ITC or SMT/TMP
produced results similar to that with P10 alone.
Combination treatments with P10 and antifungal
drugs did not further increase NO levels. Immunosuppressed, infected mice treated with CFA/IFA also
displayed an increase in NO production, albeit significantly less than with P10 or drug-treated animals.
CD11b?, Ly-6G/Ly-6C? and L3T4?Cells
in Lungs from Immunosuppressed BALB/c Mice
The phenotypic distribution of CD11b?, Ly-6G/Ly6C? and L3T4? cells was examined in the lungs of
immunosuppressed BALB/c mice, 60 days after
infection with Pb18 (Fig. 4). The tissue sections from
infected and non-immunized animals had a few
CD11b? cells that were not closely associated with

123

dispersed yeast cells (Fig. 4a). In contrast, a significant increase (p \ 0.05) in the number of CD11b?
cells in the lungs of animals immunized with P10 was
observed, with dense clustering of these cells around
rare yeast cells (Fig. 4b). These data indicate that
macrophages accumulated around P. brasiliensis
yeasts in the pulmonary tissue of immunosuppressed
mice immunized with P10 (Fig. 4b). Lungs of animals
that were not immunized showed a prominent number
of fungal cells and a faint Ly-6G/Ly-6C? cellular
staining (Fig. 4c). We can observe an intense staining
in pulmonary tissue of mice immunized with P10,
demonstrating a significant increase (p \ 0.05) in the
number of Ly-6G/Ly-6C? cells within small compact
granulomas in close proximity to rare yeast cells
(Fig. 4d). It is important to note that the monoclonal
antibody used [clone RB6-8C5 from BD Biosciences
(San Diego, CA)] reacts with a common epitope on
Ly-6G and Ly-6C that is present on neutrophils and
eosinophils. In the case of L3T4? cellular population,
pulmonary tissue of mice that were infected and not
immunized revealed a set of fungal cells and non-cell
marking was observed (Fig. 4e). The L3T4? receptor

Mycopathologia (2014) 178:177188

183

Table 1 Lung cytokine levels after 60 days of infection in immunosuppressed BALB/c mice infected with 3 9 105 yeast cells of P.
brasiliensis Pb18
Cytokine ng/ml
Treatment

Dex

Untreated

Infected

Untreated

IL-4

IL-10

IL-12

IFN-c

2.90 0.99

2.48 0.88

32.40 1.20

14.18 5.67

2.97 1.04

5.24 0.17

72.20 4.8

17.31 3.47

2.24 0.94

3.46 1.89

28.28 9.79

12.44 2.10

Infected

7.23 1.22

10.19 1.09

40.53 3.20

12.87 5.03

IFA/CFA

2.92 4.44

1.23 1.61

58.78 13.49

11.80 10.75

3.30 1.14**

1.49 1.66**

80.22 6.25*

16.94 6.62*

4.95 1.64*

1.64 1.01**

62.74 7.03*

29.49 2.02*

4.37 1.71

1.45 1.34**

83.43 3.13*

17.43 3.91*

P10
ITZ ? P10
SMT/TMP ? P10

The whole experiment was repeated twice with reproducible results

Bold data show the significant difference found between this groups and the respective control group
* Significant statistical difference (p \ 0.05) relative to immunosuppressed infected mice and not immunized
** Significant statistical difference (p \ 0.01) relative to immunosuppressed infected mice and not immunized
a

Uninfected, non-immunosuppressed, untreated and non-immunized

Infected, non-immunosuppressed, non-treatment and not immunized

Uninfected, immunosuppressed and non-immunized

Infected, immunosuppressed and non-immunized

Infected, immunosuppressed and immunized with IFA/CFA

Infected, immunosuppressed and immunized with P10

Infected, immunosuppressed immunized with P10 and treated with ITZ

Infected, immunosuppressed immunized with P10 and treated with SMT/TPM

***
25

non-Dex
Dex

NO (uM)

20

experiment, when study the lungs of mice immunized

with P10, a significant increase (p \ 0.05) in the
number of L3T4? cells that were clustered around rare
yeast cells was also detected. (Fig. 4f).

15

*
10

Immunization with P10 Minimizes the Fibrosis

in the Lung Tissue

P1
0
I
SM TZ+
P1
T/
TM
0
P+
P1
0

TM

IT

T/
SM

FA

ed

ct

A/
IF

fe
In

nf

ec
t

ed

ed
U

ni

fe
ct

ec
ni
nf
U

In

te
d

Fig. 3 Nitric oxide production in splenocyte cultures of

immunosuppressed BALB/c mice after 60 days i.t. infection
with 3 9 105 yeast of P. brasiliensis. Splenocytes are cultured
in RPMI medium in the presence of P10 peptide. NO levels are
detected using a chemiluminescence analyzer (NOATM280,
Sievers Inc., USA). *, significant difference (p B 0.05, ***
p B 0.001 relative to the infected group treated with Dex

is expressed in a subpopulation of mature T lymphocytes, including most T helper cells that exert an
important role in the host defense against fungi. In our

Reticulin and collagen fibers were analyzed in the

lungs from immunosuppressed BALB/c mice 60 days
after infection with Pb18 (Fig. 5). In mice treated with
Dex, the architecture of the lung tissue was significantly disrupted with large aggregates of yeast cells.
Collagen and reticulin fibers were abundant in the
pulmonary tissue of these animals (Fig. 5 a, c). Dextreated animals that have been immunized with P10
displayed a conserved pulmonary tissue architecture
with small compact granulomas and rare yeast cells. In
contrast with the dense fibrosis in the control groups,
the lungs of the P10 immunized animals had significantly less stainable collagen and reticulin (Fig. 5 b,d).

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Mycopathologia (2014) 178:177188

antifungal therapy (Fig. 6). In untreated controls,

100 % mortality occurred within 80 days post-infection. Animals treated with 10 mg/kg of ITC or 15 mg
sulfamethoxazole/3 mg trimethoprim/kg had significantly increased survival rates of 40-50 %, respectively. Mice immunized with P10 peptide presented a
60 % survival rate. Full protection was achieved with
the combination of P10 and ITZ or SMT/TMP with no
deaths up to a 200-day period. These results confirmed
the protective capacity of P10 peptide immunization
associated with chemotherapy.

Discussion

Fig. 4 Immunohistochemistry of CD11b?, Ly-6G/Ly-6C? and

L3T4? cell populations in pulmonary tissue from immunosuppressed and infected BALB/c mice. Tissue sections are obtained
60 days after infection with 3 9 105 yeast cells of P.
brasiliensis. a CD11b? cells in lung tissue of control group
(untreated). b CD11b? cells in lung tissues of immunized mice
with P10. c Ly-6G/Ly-6C? cells in the lung tissue of control
group, with a high number of P. brasiliensis yeasts and a few
labeled fungal cells (black arrows). d Ly-6G/Ly-6C? cells in the
lung tissue of mice immunized with P10, the white arrows show
neutrophils agglomerates. e L3T4? cells in lung tissue of the
control group and a black arrow indicated a fungal cell. f L3T4?
cells in the lung tissue of immunized mice with P10.
Diaminobenzidine (DAB) was used as the peroxidase substrate
to generate a brown-staining signal, and the sections are
counterstained with Mayer hematoxylin. Magnification 9200

P10 Immunization Increased the Survival Rates

of Immunosuppressed Mice
The survival rates of anergic BALB/c mice infected
(3x105 yeast cells/animal of Pb18) and Dex treated
were significantly increased by P10 immunization and

123

In prior work, we demonstrated that immunization

with P10 peptide induced a protective effect additive
to that of chemotherapeutic drugs, leading to a
decrease in the fungal burden and preventing the
spread of infection in the experimental model of PCM
[20, 25].
In the present study, we show the ability of P10 to
stimulate and induce a specific immune response
against P. brasiliensis in anergic animals infected with
the virulent isolate Pb18. P10 is a strong candidate for
a vaccine, leading to a predominant Th1 immune
response. This type of immune response is the most
effective against P. brasiliensis infection characterized by increased secretion of IFN-c, which stimulates
granuloma formation that contains pathogenic yeasts
[7, 20, 2527]. IFN-c is capable to activate lung
macrophages, and their increased fungicidal activity is
involved in the resistance to infection by P. brasiliensis. The absence of IFN-c, IFN-c-R or of interferon
regulatory factor-1 (IRF-1) determines the susceptibility to infection [7, 26, 27] with 100 % mortality
34 weeks after intratracheal challenge with virulent
P. brasiliensis [27].
A great deal of evidence indicates that the
response involved in the resistance to P. brasiliensis
depends mainly upon Th1 cells, while susceptibility
involves a Th2 response. The depressed cellular
immune response in particular subpopulations of
CD4? T cells has been subsequently linked to an
imbalance of cytokine regulation [28]. Previous
studies have shown that patients with the acute or
subacute form of PCM have a predominant Th2
response with high levels of specific antibodies and
increased production (in vitro) of suppressor

Mycopathologia (2014) 178:177188

185

Fig. 5 Evaluation of pulmonary fibrosis in the lungs of

immunosuppressed BALB/c mice infected with 3 9 105 yeast
cells of P. brasiliensis 60 days post-infection. a, b Massons
trichrome staining. a Lung section from an untreated group
(control), the arrow shows the accumulation of type I collagen
fibers. b Lung section from immunized with P10 group. c,
d Gomoris silver reticulin staining. c Lung section from an
untreated group, with many yeasts in the tissue, the arrow shows

an accumulation of type III collagen fibers. d Lung section from

immunized with P10 group. e, f Hematoxylineosin staining.
e Lung section from an untreated group, the arrow shows a high
number of yeasts in the tissue compromising the lung structure.
f Lung section from immunized with P10 mice, the arrow shows
a diminutive compact granuloma and a preserved pulmonary
tissue. Magnification 9100

cytokines (IL-4, IL-5, IL-10, TGF-b) and depressed

cellular immunity with low production of IFN-c and
TNF-a [34]. IL-12, IFN-c, TNF-a and IL-1b have
also been associated with resistance to PCM. The
levels of these cytokines were also high in the lung
homogenates of mice immunized with P10 and the
supernatant of cellular cultures stimulated with P10,

compared to the other groups studied. In contrast, the

P10-immunized mice displayed low levels of IL-4,
which is associated with susceptibility to PCM [34].
The pattern of cytokines released by the splenocytes
from mice immunized with P10 is consistent with a
Th1-biased T cell immune response, which is
predictive of a good clinical response.

123

186

Mycopathologia (2014) 178:177188

100

Infected
ITZ.
SMT/TMP.
P10
P10+SMT/TMP
P10+ITZ.

Survival (%)

80
60
40
20
0
0

50

100

150

200

Days

Fig. 6 Survival curves of immunosuppressed BALB/c mice i.t.

infected with 3 9 105 yeast cells of P. brasiliensis. (solid circle)
Control group (PBS). (filled square) Treated with ITZ (10 mg/
kg); (triangle) Treated with SMT/TMP (153 mg/kg); (inverted
triangle) Immunized with P10; (diamond) Immunized with P10
and treated with SMT/TMP (153 mg/kg); (open square)
Immunized with P10 and treated with ITZ (10 mg/kg); the
results are representative of two independent experiments.
p B 0.0001 for all groups compared to the control group

The effective defense against fungal infections

requires a dynamic interaction between the innate and
adaptive immune responses. The present work reports
on the induction of the immune response in mice
immunized with P10 and shows that, under experimental conditions, this peptide vaccine represents a
promising therapeutic approach for the control of
PCM.
As discussed, a significant increase in IFN-c is
associated with the induction of an activated cellular
response that is essential in host defense against P.
brasiliensis and fungal infections in general [29].
Immunization with P10 restored the capacity for
lymphoproliferation in immunosuppressed animals.
Splenocytes from anergic animals infected with Pb18
and immunized with P10, when stimulated by the
peptide in vitro showed a significantly higher lymphoproliferation rate than the splenocytes from nonimmunized animals. Lung homogenates from immunosuppressed and infected mice immunized with P10,
with or without treatment with antifungal drugs,
contained significantly higher levels of IFN-c and
IL-12 compared with the immunosuppressed and
infected mice that were not immunized (control
group) (Table 1). The splenocyte cell cultures had
increased levels of the proinflammatory cytokines IL12, TNF-a, IFN-c and IL-1b. Therefore, immunization with P10 powerfully stimulated the cellular
immune response in the immunosuppressed animals.

123

P10 immunization also led to the enhanced production of NO by macrophages. Previous studies have
shown that NO is involved in the inhibition of P.
brasiliensis conidia-to-yeast development [30]. Anergic animals infected with P. brasiliensis and immunized with P10 showed an increased production of NO
in the splenocyte culture supernatants. Such increase
could be related to the increased expression of
CD11b? cells, which were abundant in the lung tissue
of these animals. The CD11b receptor can be
expressed on macrophages, dendritic cells, natural
killer cells, microglia and B-1 cells [31]. Dendritic
cells and macrophages are part of the first line of
defense against P. brasiliensis [32].
In addition to CD11b ? cells, a significant increase
in the number of Ly-6G?/Ly-6C? and L3T4? cells
was shown in the lung tissue of immunosuppressed
animals that have been immunized with P10. Neutrophils and monocytes are Ly-6G?/Ly-6C? cells that
may be involved in the early host response against P.
brasiliensis [33]. T helper and NKT cells are L3T4?
and represent a major cell immune mechanism in host
defense in PCM.
A pro-inflammatory response is important to contain
the infection and the spread of the fungus in the host, but
the deleterious effects of an exacerbated immune
response have to be controlled. Pulmonary fibrosis is a
severe and progressive sequel of PCM. Development of
pulmonary fibrosis is probably attributed to a prolonged
inflammatory stimulus, which is common in PCM [34].
It is noteworthy that animals immunized with P10
showed a decrease in pulmonary fibrosis. A likely
explanation is the remarkable reduction of fungal
burden in the lungs of immunized animals, which leads
to decreased stimulation and less pulmonary fibrosis.
Detection of IFN-c, TNF-a, IL-1b and IL-18 in the
lungs (data not shown), indicated restoration of
cellular immunity in anergic animals immunized with
P10. In PCM, TNF-a is directly related to the
formation of granulomas [35], and together with IL12 and IL-18, potentiates the fungicidal activity of
macrophages.
Evaluation of cytokine responses as well as their
effects on cellular populations is increasingly important for understanding the global immunomodulatory
processes that occur during infection. Recently, new
subpopulations of effectors CD4? T cells (Th9, Th17
and Th22 cells) have been identified as important
mediators in response to fungal infections [35 ].

Mycopathologia (2014) 178:177188

However, further studies are needed to better understand the resistance mechanisms to pulmonary PCM in
different patient populations.
As compared to 100 % of immunosuppressed and
infected animals that died within 80 days after infection with virulent P. brasiliensis Pb18, those immunized with P10 and treated with sulfamethoxazole/
trimethoprim or ITC were fully protected as all
animals in these treatment groups were alive at the
end of our experiment (200 days).
Currently, treatment of PCM depends on the
severity of the disease, type of drug utilized and the
time of use. Treatment of PCM is often compromised
due to the toxicity of the protracted antifungal therapy
required and relapses due to fungal resistance have
been reported [13]. Presently, we show that peptide
P10 immunization restores specific immunity against
P. brasiliensis even in animals subjected to immunosuppressive treatment with a synthetic corticosteroid
(Dex) in an experimental model of PCM. Complete
protection against the fungal infection without fibrotic
side effects was achieved using a combined approach
of chemotherapeutic drugs and P10 immunization.
The use of P10 as an adjuvant to antifungal
treatment is a promising tool in the treatment of
PCM and this modality also can be considered for the
prevention of relapses [20]. Combined treatment could
be important even in cases of clinical resistance to
azoles and sulfamethoxazole/trimethoprim. The present data strongly suggest that immunization with P10
peptide, even in the setting of immunosuppression, has
significant therapeutic benefits in experimental PCM,
which further supports pursuing clinical trials with the
peptide as a vaccine candidate in human PCM.
Acknowledgments This work was supported by grants
2007/58750-4, 2011/17267-4 and 2010/51423-0 from Fundacao
de Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP) and
Conselho Nacional de Desenvolvimento Cientfico e Tecnologico
(CNPq). We acknowledge the valuable technical assistance of the
Laboratory the Immunohistochemistry of Department of
Anatomy, School of Veterinary Medicine and Animal Science,
University of Sao Paulo, and of Carlos da Silva and the animal
facility of Department of Microbiology, University of Sao Paulo.

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