Immunization with P10 Peptide Increases Specific Immunityand Protects Immunosuppressed BALB/c Mice Infectedwith 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

� 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 intra-

tracheal challenge with virulent P. brasiliensis. Pre-

viously, 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

increased nitric oxide (NO) production and induced

high levels of IFN-c, IL-1b and IL-12. Furthermore,

significantly increased concentrations of pro-inflam-

matory cytokines were also observed in lung homog-

enates 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 paracoccidioido-

mycosis (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

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

123

Mycopathologia

DOI 10.1007/s11046-014-9801-1

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 immu-

nity, the absence of delayed-type hypersensitivity

reactions and high mortality rate. The chronic form

affects mainly adult males of 30–50 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 macro-

phages 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 facil-

itating 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 delayed-

type 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 impor-

tantly, most of the human DR antigens promiscuously

present P10 and neighbor peptides [12].

Treatment of PCM requires intensive and pro-

longed antifungal chemotherapy. Treatment with

sulfonamides (either sulfamethoxazole or sulfadia-

zine) combined with trimethoprim, amphotericin B or

an azole is typically administered for 2–6 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

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 dis-

ease 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 modula-

tion 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 preserva-

tion 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, 6–8-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 ICB–USP 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

123

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.

Fungal Strain

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 7–10 days of growth, yeast cells were

cultivated in modified McVeigh-Morton medium at

37 �C for 5–7 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 anesthe-

tized i.p. with 200 ll of a solution containing 80 mg/

kg ketamine and 10 mg/kg of xylazine (both from

Uniao Quımica Farmaceutica, Brazil). After approx-

imately 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 (6–8 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

immunization was subcutaneous with P10 in complete

Freund’s adjuvant (CFA) followed by i.p. immuniza-

tion of the peptide in incomplete Freund’s adjuvant

(IFA). Control mice were injected with CFA and IFA

alone without the peptide.

Treatment with Antifungal Drugs

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.

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 immu-

nosuppressed with Dex phosphate; infected, immuno-

suppressed 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), immunosup-

pressed 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 FBS-

free RPMI, counted, added to 96-well plates at

4 9 105 cells/well and final volume of 200 ll. The

Mycopathologia

123

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). Naıve splenocytes

from the sham group were used as negative control. To

determine cell viability/proliferation, MTT (1 mg/ml,

Thiazolyl Blue Tetrazolium Bromide–Sigma, St

Louis) was added to each well during the final 4 h of

culture [22]. The reaction was terminated with 100 ll/

well of isopropanol–HCl 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 chemilumines-

cence analyzer (NOATM280, Sievers Inc., USA). A

calibration curve was set using sodium nitrate stan-

dards. Using the NOATM280 analyzer, nitrate was

reduced to NO with vanadium (III) at 90 �C and the

NO formed was detected by gaseous phase chemilu-

minescence 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

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 Lab-

oratories, Burlingame, CA, USA) for 1 h at room

temperature. Chromogen 3,3-diaminobenzidine tetra-

hydrocloride (DAB; Sigma-Aldrich, St. Louis, MO,

USA) was used to localize peroxidase in tissue sections.

Finally, the slides were counterstained with Mayer’s

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 Gomori’s

silver reticulin–stain to assess the changes occurring in

the organization of reticulin fibers (collagen III), and

Masson’s trichrome–stain 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 immuni-

zation 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

123

Statistical Analysis

Statistics was performed using GraphPad Prism5

software (San Diego, CA). The results were expressed

as means and SD. The nonparametric Tukey’s hon-

estly significant difference test was employed. p values

of B0.05 indicated statistical significance. In the case

of the survival curve, the Log-rank (Mantel–Cox) test

with p values of B0.0001 was used to indicate

statistical significance. Unpaired Student’s t test with

Welch’s correction (two tailed) was used for the

comparison of two groups when the data met the

assumptions of the t tests.

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 immu-

nized 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-immunosup-

pressed mice. In both positive control groups, we

observed that splenocyte proliferation was similar to

that achieved in immunosuppressed and P10-stimu-

lated 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

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 subse-

quently infected and immunized with P10 peptide

produced significantly increased amounts of IL-12 and

IFN-c while concomitantly having significant reduc-

tions 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 prolif-

eration results shown in Fig. 1.

Non-s

timula

ted

ConA

Infe

cted

Infe

cted

IFA/C

FAIT

Z

SMT/T

MP

P10

P10+I

TZ

P10+S

MT/T

MP

0.0

0.1

0.2

0.3

0.4

0.5Dexnon-Dex

D.O

. 590

* *

Fig. 1 P10 immunization of splenocytes from Dex-immuno-

suppressed 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

Mycopathologia

123

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. Immuno-

suppressed, infected mice treated with CFA/IFA also

displayed an increase in NO production, albeit signif-

icantly 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/Ly-

6C? 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

dispersed yeast cells (Fig. 4a). In contrast, a signifi-

cant 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

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. (non-

Dex): 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)

Mycopathologia

123

is expressed in a subpopulation of mature T lympho-

cytes, including most T helper cells that exert an

important role in the host defense against fungi. In our

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).

Immunization with P10 Minimizes the Fibrosis

in the Lung Tissue

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 signifi-

cantly disrupted with large aggregates of yeast cells.

Collagen and reticulin fibers were abundant in the

pulmonary tissue of these animals (Fig. 5 a, c). Dex-

treated 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 signifi-

cantly less stainable collagen and reticulin (Fig. 5 b,d).

Uninfe

cted

Infe

cted

Uninfe

cted

Infe

cted

IFA/C

FAIT

Z

SMT/T

MP

P10

ITZ+P

10

SMT/T

MP+P

100

5

10

15

20

25

*

***

non-Dex

Dex

NO

(uM

)

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

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 IL-4 IL-10 IL-12 IFN-c

aUntreated - 2.90 ± 0.99 2.48 ± 0.88 32.40 ± 1.20 14.18 ± 5.67bInfected - 2.97 ± 1.04 5.24 ± 0.17 72.20 ± 4.8 17.31 ± 3.47cUntreated ? 2.24 ± 0.94 3.46 ± 1.89 28.28 ± 9.79 12.44 ± 2.10dInfected ? 7.23 ± 1.22 10.19 ± 1.09 40.53 ± 3.20 12.87 ± 5.03eIFA/CFA ? 2.92 ± 4.44 1.23 ± 1.61 58.78 ± 13.49 11.80 ± 10.75fP10 ? 3.30 – 1.14** 1.49 – 1.66** 80.22 – 6.25* 16.94 – 6.62*gITZ ? P10 ? 4.95 – 1.64* 1.64 – 1.01** 62.74 – 7.03* 29.49 – 2.02*hSMT/TMP ? P10 ? 4.37 ± 1.71 1.45 – 1.34** 83.43 – 3.13* 17.43 – 3.91*

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 immunizeda Uninfected, non-immunosuppressed, untreated and non-immunizedb Infected, non-immunosuppressed, non-treatment and not immunizedc Uninfected, immunosuppressed and non-immunizedd Infected, immunosuppressed and non-immunizede Infected, immunosuppressed and immunized with IFA/CFAf Infected, immunosuppressed and immunized with P10g Infected, immunosuppressed immunized with P10 and treated with ITZh Infected, immunosuppressed immunized with P10 and treated with SMT/TPM

Mycopathologia

123

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

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

100 % mortality occurred within 80 days post-infec-

tion. Animals treated with 10 mg/kg of ITC or 15 mg

sulfamethoxazole/3 mg trimethoprim/kg had signifi-

cantly increased survival rates of 40-50 %, respec-

tively. 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

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 character-

ized by increased secretion of IFN-c, which stimulates

granuloma formation that contains pathogenic yeasts

[7, 20, 25–27]. IFN-c is capable to activate lung

macrophages, and their increased fungicidal activity is

involved in the resistance to infection by P. brasili-

ensis. The absence of IFN-c, IFN-c-R or of interferon

regulatory factor-1 (IRF-1) determines the suscepti-

bility to infection [7, 26, 27] with 100 % mortality

3–4 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

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

L3T4? cell populations in pulmonary tissue from immunosup-

pressed 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

Mycopathologia

123

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.

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 Masson’s

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 Gomori’s 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 Hematoxylin–eosin 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

Mycopathologia

123

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 experi-

mental 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 lym-

phoproliferation rate than the splenocytes from non-

immunized animals. Lung homogenates from immu-

nosuppressed 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 IL-

12, TNF-a, IFN-c and IL-1b. Therefore, immuniza-

tion with P10 powerfully stimulated the cellular

immune response in the immunosuppressed animals.

P10 immunization also led to the enhanced pro-

duction of NO by macrophages. Previous studies have

shown that NO is involved in the inhibition of P.

brasiliensis conidia-to-yeast development [30]. Aner-

gic animals infected with P. brasiliensis and immu-

nized 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. Neutro-

phils 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 IL-

12 and IL-18, potentiates the fungicidal activity of

macrophages.

Evaluation of cytokine responses as well as their

effects on cellular populations is increasingly impor-

tant 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 ].

0 50 100 150 2000

20

40

60

80

100Infected

ITZ.

SMT/TMP.

P10

P10+SMT/TMP

P10+ITZ.

Days

Su

rviv

al (

%)

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 (15–3 mg/kg); (inverted

triangle) Immunized with P10; (diamond) Immunized with P10

and treated with SMT/TMP (15–3 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

Mycopathologia

123

However, further studies are needed to better under-

stand 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 infec-

tion with virulent P. brasiliensis Pb18, those immu-

nized 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 immuno-

suppressive 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 pres-

ent 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 Cientıfico 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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