Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental Paracoccidioides lutzii

Q5

Q1

Q2

Q3

+ MODEL MICINF4177_proof ■ 27 August 2014 ■ 1/8

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263

Microbes and Infection xx (2014) 1e8www.elsevier.com/locate/micinf

6465666768697071727374757677787980818283848586

Original article

Monoclonal antibodies to heat shock protein 60 induce a protective immuneresponse against experimental Paracoccidioides lutzii

Luciana Thomaz a, Joshua D. Nosanchuk b,c, Diego C.P. Rossi a, Luiz R. Travassos e,Carlos P. Taborda a,d,*

a Institute of Biomedical Sciences, Department of Microbiology, Brazilb Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

c Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USAd Laboratory of Medical Mycology IMTSP- LIM53 University of S~ao Paulo, S~ao Paulo, Brazil

e Department of Microbiology, Immunology and Parasitology, Federal University of S~ao Paulo, S~ao Paulo, Brazil

Received 16 September 2013; accepted 6 August 2014

878889
90 Abstract 919293949596979899
100101102103104105106107108109110

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and lessfrequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration ofsystemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches totreatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) fromHistoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that theHsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs toHsp60 before infection reduced the pulmonary fungal burden as compared to mice treated with irrelevant mAb. Hence, mAbs raised to H.capsulatum Hsp60 are protective against P. lutzii, including mAb 7B6 which was non-protective against H. capsulatum, suggesting differences intheir capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungusmay be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb isbeneficial or harmful.© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Paracoccidioides lutzii; Hsp60; Monoclonal antibody

111112113114115116117118119

1. Introduction

Paracoccidioidomycosis (PCM) is caused by a complexgroup of fungi within the Paracoccidioides genus comprisingfour distinct phylogenetic lineages known as PS2, PS3, S1 andPb01-like [1]. Pb01 is morphologically and genetically distinctfrom the other lineages, which has led to its designation as

* Corresponding author. Institute of Biomedical Sciences, Department of

Microbiology, Av. Prof. Lineu Prestes, 1351 e sala 247, 05508900 S~ao Paulo,

Brazil. Tel.: þ55 11 3091 7351.

E-mail address: [email protected] (L. Thomaz).

Please cite this article in press as: Thomaz L, et al., Monoclonal antibodies to hea

Paracoccidioides lutzii, Microbes and Infection (2014), http://dx.doi.org/10.1016/

120121122123124125

http://dx.doi.org/10.1016/j.micinf.2014.08.004

1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights

Paracoccidioides lutzii [2], while the others are speciated asParacoccidioides brasiliensis. However, the pathogenesis anddisease manifestations of P. brasiliensis and P. lutzii areindistinguishable at present. PCM is initiated by the inhalationof airborne conidia released by the filamentous forms of thesefungi that are present in soil under certain conditions. Theseconidia reach the pulmonary alveoli and transition into yeastforms [3]. The disease can develop rapidly after acquisition ofthe conidia or manifest years after the infection. There are twomain clinical forms that patients develop: acute/subacute orchronic. The acute/subacute form is more common amongchildren and young adults, and it is characterized by

t shock protein 60 induce a protective immune response against experimental

j.micinf.2014.08.004

126127128reserved.

mailto:[email protected]




2 L. Thomaz et al. / Microbes and Infection xx (2014) 1e8

MICINF4177_proof ■ 27 August 2014 ■ 2/8

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129

lymphadenopathy associated with hepatosplenomegaly as wellas cutaneous, intestinal and bone lesions [4]. The chronic formtypically affects adults, and it is characterized by pulmonarylesions, often associated with oral, nasal, or laryngeal ulcers.There is both an increased incidence and enhanced severity ofPCM in patients co-infected with HIV [5,6].

In the absence of systemic antimicrobial therapy, PCM isuniformly progressive. Standard treatment regimens utilizesulfonamides, amphotericin B or azoles, and therapy durationis from 2 months to several years, depending on diseaseseverity and host responsiveness [4]. Despite protractedtreatment approaches, relapses are common. Regarding hostresponses, there is ample evidence that the activation ofcellular immunity is the most effective mechanism to controlexperimental and human PCM, although both innate immuneresponses and adaptive immunity are important for manifest-ing effective protection (reviewed in Ref. [7]). The role of thehumoral immune response in PCM is controversial especiallyas patients with acute and sub acute forms of the disease havehigh antibody titers [7]. However, for several related fungalpathogens there is considerable evidence that the administra-tion of monoclonal antibodies (mAbs) can modify the courseof experimental diseases. Protective mAbs have been suc-cessfully identified against some important fungi, such asAspergillus fumigatus, Candida albicans, Cryptococcus neo-formans, and Histoplasma capsulatum (reviewed in Refs.[7,8]). Moreover, 10 years ago a mAb to a 70 kDa glycopro-tein was shown to modify experimental PCM [9]. Since thisobservation, several groups have begun to explore the impactof mAbs targeting Paracoccidioides complex antigens.

Heat shock proteins (Hsp) have emerged as a potentialtarget for therapeutic mAbs as they are conserved immuno-dominant antigens that can evoke cell mediated and humoralresponses during infection [10e12]. H. capsulatum is a relateddimorphic fungus that displays a 62 kDa surface form of Hsp(Hsp60) that is involved in the adhesion of the parasitic yeastform to host macrophages via interaction with CD18 integrinreceptors [13]. MAbs targeting H. capsulatum Hsp60 modifythe pathogenesis of murine histoplasmosis, and the protectiveefficacy of individual mAbs in histoplasmosis appears tocorrespond to the Ig subclass [14]. In the Histoplasma model,mAb 7B6, an IgG2b, increased tissue inflammation, enhancedintracellular yeast growth and was not protective, whereasIgG1 and IgG2a mAbs reduced inflammation, impeded intra-cellular replication, and prolonged the survival of mice [14].

Paracoccidioides spp. also expresses Hsp60 and the proteinis produced in large amounts in response to rapid increases intemperature and other types of stress. The greater expressionof Hsp60 in yeast cells compared to mycelium suggests thatthis protein could be necessary for survival in host conditionsand that it may have a role in morphogenesis [15,16]. In thepresent study, we examined the impact of 2 mAbs generatedagainst Hsp60 from H. capsulatum, one characterized as non-protective (mAb 7B6, IgG2b) and another as protective(mAb 4E12, IgG2a), in a validated experimental para-coccidioidomycosis model.


Paracoccidioides lutzii, Microbes and Infection (2014), http://dx.doi.org/10.1016

2. Materials and methods

2.1. Animals

BALB/c mice (6e8 weeks female) were bred at the Insti-tute of Biomedical Sciences of University of S~ao Paulo (USP),Brazil, animal facility, under specific pathogen-free condi-tions. The procedures involving animals and their care wereconducted according to the local ethics committee and inter-national rules. All procedures were approved by the EthicsCommittee on the Use of Animals (CEUA) of USP.

2.2. Fungal strain and growth conditions

Virulent P. lutzii strain Pb01 yeast cells were grown inSabouraud liquid medium at 37 �C with shaking and wereused after 5 days of growth. The fungal cells were washed inphosphate-buffered saline, pH 7.2 and counted in a hemocy-tometer. Cell viability was determined by staining with Trypanblue (Sigma, St.Louis, MO) and was higher than 90% [17].

2.3. MAbs

The mAbs against Hsp60 e 4E12 (IgG2a) and 7B6 (IgG2b)- from H. capsulatum were previously characterized [14]. ThemAbs were purified using a protein A/G affinity chromatog-raphy (Pierce, Rockland, IL) following the manufacturer'sinstructions. MAb concentrations were determined by Nano-drop 100 (Thermo Scientific, Wilmington, DE).

2.4. Phagocytosis assay

Primary peritoneal Macrophages from BALB/c mice werecultivated in a medium containing 10% heat-inactivated fetalcalf serum, 10% NCTC-109 medium (Life Technologies,Grand Island, NY), nonessential amino acids (Life Technol-ogies) and 1% Pen-Strep. Macrophage cells were plated on 96-well tissue culture plates at a final density of 1.6 � 105 cells/well and incubated at 37 �C for 24 h with IFN-g. P. lutzii yeastcells were collected after 5 days of growth, washed three timeswith PBS and incubated with or without mAb for 24 h. ThemAbs were tested at two concentrations, 10 and 50 mg/ml. Anirrelevant IgG (IgG2a) mAb [18] was used as an additionalcontrol. P. lutzii cells were added at 5:1 macrophage to fungalcell ratio and incubated for 12 h. The cells were then washedseveral times with PBS, and then with 0.15 M a-mannopyr-anoside (Sigma) to remove extracellular yeast. Samples werefixed with cold absolute methanol, and then stained with a 1/20 solution of Giemsa (Sigma). The number of phagocytosedyeast was determined by light microscopy at � 400 magnifi-cation. The phagocytic index (PI) is defined as PI ¼ P � F,where P is the percentage of macrophages with internalizedyeast and F is the average number of yeast cells per macro-phage. Experiments were carried out in triplicate. Thenumbers of macrophage and yeast cells were recorded for eachfield, and at least 200 macrophages were counted.


/j.micinf.2014.08.004

130

3L. Thomaz et al. / Microbes and Infection xx (2014) 1e8


1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66
2.5. Immunofluorescence 6768697071727374757677787980818283848586
P. lutzii yeast cells were grown as described and pelleted bycentrifugation at 500 � g for 5 min. The cells were washedwith PBS, and 5 � 106 cells were suspended in 1% PBS-BSAwith 100 mg/mL of mAbs anti-Hsp60, 4E12 and 7B6 orirrelevant mAb overnight at 4 �C. The cells were then washedwith 1% PBS-BSA three times and suspended in 1% PBS-BSA with FITC-conjugated goat anti-mouse IgG secondaryantibody (BD-Pharmigen) overnight at 4 �C. The sampleswere then washed five times with 1% PBS-BSA and sus-pended for 10 min in 1% Calcofluor (Sigma) in PBS buffer atroom temperature. The cells were then washed three timeswith PBS and suspended in 30 ml of N-propyl-gallate (Sigma)in glycerol (Sigma). Aliquots were placed on glass slides withcover slips and viewed by light and immunofluorescence mi-croscopy EVOS microscope (Advanced Microscopy Group,Bothel, WA) using the AMG camera.

8788
2.6. Intratracheal infection of BALB/c mice 8990919293949596979899
BALB/c mice were each inoculated intratracheally (IT)with 50 ml of 1 � 106 yeast cells of Pb01. Mice were anes-thetized intraperitoneally (IP) with 200 ml of a solution of80 mg/kg ketamine/kg of body weight and 10 mg/kg ofxylazine (Uni~ao Química Farmaceutica, Brazil). Approxi-mately 10 min after anesthetization, the neck was hyper-extended, the trachea exposed at the level of thyroid, and theyeast cells were introduced via a 26-gauge needle. The in-cisions were sutured with 5e0 silk.

100101102
2.7. Fungal burden in organs of infected mice
103104105106107108109110111112113

Mice were euthanized 15 days after IT infection. The lungs,liver and spleen were aseptically removed and sectioned.Portions of the lungs were weighed and then homogenized in1 ml of PBS. A 100 ml aliquot from each suspension wasinoculated on brain-heart infusion (BHI) agar plates, supple-mented with 4% fetal calf serum (Vitrocell, Brazil), 5% spentP. brasiliensis (strain 192) culture medium, streptomycin/penicillin 10 IU/ml (Sigma). Colony countings were madeafter 10 days of incubation at 37 �C.

114115
2.8. Histology 116117118119120121122
Sections of excised lungs were fixed in 10% bufferedformalin, embedded in paraffin. The sections were stainedwith Grocott silver or Hematoxylin and Eosin (HE) andexamined microscopically at 40 � (Optiphot-2; Nikon, Tokyo,Japan).

123124
2.9. Cytokine analysis 125126127128129130
Weighed sections of lung were homogenized in 1 ml ofPBS in the presence of protease inhibitors (Sigma) to preventdegradation of cytokines by tissue proteases, centrifuged, andthe supernatants frozen at e 80 �C until tested in accordance



with the manufacturer's instructions. The supernatants wereassayed for IL-4, IL-10, IL-12 and IFN-g using ELISA kits(BD PharMingen, San Diego, CA). The detections limits ofsuch assays were as follows: 7.8 pg/ml for IL-4; 31.25 pg/mlfor IL-10; 15.6 pg/ml for IL-12 and 15.6 pg/ml for IFN-g,according to the manufacturer.

2.10. Statistical analysis

Statistical analyses were performed using GraphPad Prismversion 5.00 for Windows (GraphPad Software, San Diego,CA). Statistical comparisons were made by analysis of vari-ance (one-way ANOVA) followed by a Turkey-Kramer post-test. ( p) values of p < 0.05 indicated statistical significance. A95% confidence interval was determined in all experiments.

3. Results

3.1. Immunofluorescence

To validate the binding of mAbs 4E12 and 7B6 to P. lutziiHsp60, we assessed the interactions of the mAbs with yeastcells by fluorescence microscopy (Fig. 1). MAb 4E12 labeledalmost all P. lutzii yeast cells diffusely at the cell wall, whereasmAb 7B6 labeled yeast cells less robustly. In contrast, theirrelevant mAb did not bind the fungal cells.

3.2. Phagocytosis assay

We analyzed the effects of mAbs 4E12 (IgG2a) and 7B6(IgG2b), on the phagocytosis of P. lutzii by primary macro-phages from BALB/c mice. Ten and 50 mg per milliliter ofeither 7B6 or 4E12 significantly enhanced the phagocytosis ofyeast cells compared to irrelevant mAb, p < 0.0001 (Fig. 2).

3.3. Fungal burden

To evaluate whether the administration of mAbs mediatedprotection in BALB/c mice that received 1 mg/animal ofmAbs 24 h before IT infection with P. lutzii, we examined thelungs, spleens and livers 15 days after infection. Both groupsthat received Hsp60-binding mAbs had significant reductionsin the number of pulmonary CFUs compared with the controlgroup treated with irrelevant mAb ( p < 0.05) (Fig. 3). How-ever, MAb 4E12 was significantly more effective at reducingthe pulmonary fungal burden compared to mAB 7B6( p < 0.005). No CFUs were recovered from the spleens andlivers of any mice.

3.4. Lung histology

Distinct differences in histology were observed between themice that received Hsp60-binding mAbs relative to controls.In the control groups (Figs. 4 and 5A), histological reviewshowed confluent foci of inflammation granulomas throughoutthe lungs and a high resolution study revealed the predomi-nance of neutrophils as well as numerous replicating fungal


j.micinf.2014.08.004

Fig. 1. Hsp60-binding MAbs bind P. lutzii yeast cells. Analyzes by brightfield and fluorescence microscopy of yeast cells of P. lutzii 01 incubated with irrelevant

mAb (A and B) or mAbs 4E12 (C and D) and 7B6 (E and F). Green fluorescence represents binding by antibody and blue fluorescence labeling by calcoflour. Scale

bars, 50 mm.

Fig. 2. mAb to Hsp60 modify phagocytosis of P. lutzii by peritoneal mac-

rophages. Phagocytosis of P. lutzii by primary peritoneal macrophages from

BALB/c mice after 24 h in the presence of two concentrations of mAbs against

Hsp60 compared to phagocytosis of yeast cells with irrelevant mAb. Each

experiment was done in triplicate. ***, p < 0.0001, comparing Hsp-binding

mAb to control mAb.

Fig. 3. Hsp60-binding mAbs reduce P. lutzii fungal burdens in the lungs of

infected mice. Lung CFU from mice 15 days after infection with P. lutzii. The

animals had received either Hsp60-binding mAb (7B6 or 4E12) or irrelevant

mAb 24 h before IT infection with P. lutzii 01. Each bar represents the average

of two similar experiments. *, p < 0.05 and **, p < 0.005, significant dif-

ference relative to the irrelevant control.


Please cite this article in press as: Thomaz L, et al., Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental

Paracoccidioides lutzii, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.004


1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

Fig. 4. Gomori�s methenamine silver staining reveals that Hsp60-binding mAbs reduce the number of P. lutzii in the lungs of infected mice. Gomori�s

methenamine silver staining of lung sections from mice 15 days after infection with P. lutzii. The animals had received either Hsp60-binding MAb (7B6 or 4E12)

or irrelevant mAb 24 h before IT infection with P. lutzii 01. (A) irrelevant mAb, (B) mAb 7B6 and (C) mAb 4E12. � 40 magnification. Black arrows point to yeast

cells.



1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

6667686970717273747576777879808182838485868788899091929394959697

cells. There were few areas of intact lung architecture in thecontrol infected mice. In contrast, the pathological review ofthe lungs of mice treated with either mAbs 7B6 (Figs. 4 and5B) or 4E12 (Figs. 4 and 5C) revealed pulmonary lesionsconsisting largely of epithelioid pauciparasitic granulomas.Fungal cells were present in mice treated with mAb 7B6 (Figs.4 and 5B), albeit at lower amount. Also of note, mice treatedwith mAb 4E12 had lower numbers of yeast cells compared tocontrols or mAb 7B6 treated mice, consistent with the CFUresults shown in Fig. 3, and the mice that received mAb 4E12displayed early organization of granulomas surrounding thefungal cells (Figs. 4 and 5C). Additionally, the mAb 4E12-treated mice had large areas of histologically normal lungtissue (Figs. 4 and 5).

9899
3.5. Cytokine production
100101102103104105106107108109110111112113114115

Alterations in cytokine levels were detected in the lungtissues of IT infected mice treated with MAb 7B6 and 4E12compared to mice that received irrelevant mAb (Fig. 6). ThemAb 4E12 treated and infected mice had the highest levels ofIFN-g ( p < 0.01) and IL-12p70 ( p < 0.05) and lowest levelsof IL-4 ( p < 0.01) and IL-10 ( p < 0.01). MAb 7B6 treatmentalso reduced IL-4 ( p ¼ 0.049) and IL10 ( p < 0.05) levelscompared to the irrelevant antibody, although to a lesser extentthan those achieved with mAb 4E12. Similarly, IFN-g wasincreased in mAb 7B6 treated mice ( p ¼ 0.018), but IL-12p70was not. Interestingly, mAb 7B6 significantly reduced IL-12p40 levels relative to control antibody ( p < 0.05), but notin comparison with mAb 4E12 ( p > 0.05). Therefore the level

Fig. 5. Hsp60-binding mAbs reduce the number of P. lutzii in the lungs of infec

after infection with P. lutzii. The animals had received either Hsp60-binding MAb (

irrelevant mAb, (B) mAb 7B6 and (C) mAb 4E12. � 40 magnification. Black arr



of IL-4 decreased by 37% with 7B6 and 73% with 4E12 whencompared with the irrelevant antibody; for IL-10 the levelswere almost 52% lower with 7B6 and 69% with 4E12; for IL-12p40 the concentrations were 56% lower with 7B6 and 37%with 4E12; the IFN-g levels increased 260% with 7B6 and~450% with 4E12; and the IL-12p70 concentrations increased32% with 7B6 and 143% with 4E12. In conclusion, the IgG2aand IgG2b mAbs to Hsp60 induced a Th1-biased response,with a concomitant reduction in fungal burden.

4. Discussion

PCM is the predominant human systemic mycosis in LatinAmerica [7] caused by a dimorphic fungus. While histoplas-mosis is the prevalent systemic fungal dimorphic mycosis inCentral and North American countries [19], PCM is predom-inantly found in South America [20]. Approximately 80% ofPCM cases in Latin America have been reported in Brazil,primarily in the states of S~ao Paulo, Paran�a, Rio Grande doSul, Goi�as, Rio de Janeiro, and Rondonia [21,22]. PCM, his-toplasmosis, coccidioidomycosis, and blastomycosis sharesome important attributes, including dimorphism, immuno-dominant antigens and several clinical and pathologicalcharacteristics [23]. Although antifungal therapy is the uni-versally used treatment of PCM, the passive transfer of spe-cific mAb may boost cellular immune responses and augmentthe protective effect of chemotherapy [7].

In 2003, two IgG1 mAbs to P. brasiliensis 70 kDa glyco-protein, which is recognized by 96% sera from PCM patients,were highly protective in experimental murine PCM when

ted mice. Hematoxylin and Eosin staining of lung sections from mice 15 days

7B6 or 4E12) or irrelevant mAb, 24 h before IT infection with P. lutzii 01. (A)

ows point to yeast cells.


j.micinf.2014.08.004

116117118119120121122123124125126127128129130

Fig. 6. Hsp60-binding mAbs alters the cytokine response in the lungs of P. lutzii infected mice. Cytokine levels in the lungs of mice in response to the passive

transfer of mAbs 7B6, 4E12 and irrelevant 24 h before IT infection with P. lutzii. The levels of IL-4, IL-10, IL-12p40, IL-12p70 and IFN-g were determined by

ELISA. Each bar represents the average of two similar experiments. *, p < 0.05, **, p < 0.005 and ***, p < 0.0001, significant difference relative to the irrelevant

control.



1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

administered at the time of infection [9]. The histology of lungsections from mice infected with P. brasiliensis and treatedwith mAb revealed significantly reduced fungal burdens and aprotective cellular response characterized by granuloma for-mation. The main diagnostic antigen for P. brasiliensis, thegp43, which is recognized by virtually 100% of patients withPCM [24], has also been explored as a target for antibodytherapy. Previous data from our group demonstrated that thepassive administration of mAb 3E (IgG2b) to gp43 before andafter intratracheal infection or intravenous infection reducedfungal burden and decreased pulmonary infection, whereasmAb 32H (IgG2a) was not protective [25]. We also investi-gated the effect of mAbs to gp43 on phagocytosis using pri-mary macrophages (lung and peritoneal) and cell lines(J774.16 and MH-S). Yeast cells opsonized with protectivemAbs were internalized at least twofold more than yeast cellsin the absence of antibody or opsonized by non-protectivemAbs. The results with primary macrophages and cell lineswere similar [25].



In addition to belonging to different isotypes, these gp43-binding mAbs react with distinct epitopes. It should benoted that patients infected with P. lutzii usually have negativeserological responses in gp43-antigen based assays [26]. AnIgG2a mAb to P. brasiliensis glycosphingolipids reduced thegrowth and morphogenesis of P. brasiliensis, H. capsulatum,and Sporothrix schenckii, in vitro [27]. IgM and IgG2a mAbsto gp75, a P. brasiliensis protein with phosphatase activity,inhibited fungal growth in vitro and passive immunizationusing either mAb prior to and during infection reduced pul-monary fungal burdens and the lungs of the treated mice werehistologically normal after 45 days infection [28].

Given the high level of conservation among the dimorphicfungal Hsp family, and particularly between H. capsulatumand the P. brasiliensis Hsp60 complex [23], we tested theeffects of mAbs generated to H. capsulatum Hsp60 in anexperimental P. lutzii infection model. In murine histoplas-mosis, the IgG2a mAb prolonged the survival of mice asso-ciated to a strong Th-1 response while the IgG2b mAb


/j.micinf.2014.08.004

Q4



1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

exacerbated disease and polarized the host reaction toward aTh-2 response [14].

In the present work, we showed that mAbs 4E12 and 7B6were biologically active against P. lutzii. Both isotypesenhanced P. lutzii phagocytosis in vitro. Passive administrationof the mAbs prior to IT infection of P. lutzii in mice signifi-cantly reduced the fungal burden in pulmonary tissue. Thiscontrasts with the disease enhancing effect of mAb 7B6 inhistoplasmosis. Moreover, as assessed by histology, bothmAbs reduced the pulmonary tissue damage due to PCM,albeit the protective effect of mAb 4E12 was significantlymore pronounced. This may be attributed to the more robustbinding, as determined by fluorescence assays, of mAb 4E12to P. lutzii yeast cells relative to that observed with mAb 7B6.Additionally, the formation of compact granulomas in themice treated with mAb 4E12 can be associated with the robustTh-1 biased response identified by the cytokine assays.Although mAb 7B6 was associated with a Th-2 response inhistoplasmosis, we found that this mAb contributed to a Th-1biased response in PCM. Interestingly, mice treated with mAb7B6 had significantly lower levels of IL-12p40 than controlanimals. IL-12p40 may block IL-12p70 associated responses[29], so that a reduction in IL-12p40 may be a protectiveresponse even in face of a lower induction of IL-12p70 bymAb 7B6 relative to MAb 4E12. The role of IFN-gammamediating activated macrophages in paracoccidioidomycosishas been well documented [reviewed in 25]. Murine peritonealmacrophages and cell line activated by IFN-gamma showenhanced fungicidal activity against P. brasiliensis [25].However, we showed here a similar mechanism against P.lutzii in which mAbs 7B6 and 4E12 significantly enhance IFN-gamma production. In conclusion, we demonstrate the pro-tective efficacy of mAbs raised to H. capsulatum Hsp60 in amurine PCM model. Moreover, we have found significantdifferences in the infection outcomes as MAb 7B6 exacerbateshistoplasmosis while it is protective in PCM. This strikingobservation suggests that key differences may happen in thebiology of these fungal pathogens or in the manner in whichmammalian hosts respond to their presence. Future studieswill be undertaken to explore the mechanisms behind thesedifferences.

Acknowledgments

The present work was supported by FAPESP grant 2007/53175-1, 2011/17267-4, 2013/18655-3. CPT and LRT arecareer research fellows of the CNPq. We thank Allan J.Guimar~aes for development of the hybridomas 4E12 and 7B6.

References

[1] Matute DR, McEwen JG, Puccia R, Montes BA, San-Blas G, Bagagli E,

et al. Cryptic speciation and recombination in the fungus Para-

coccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol

2006;23:65e73.

[2] Teixeira MM, Theodoro RC, de Carvalho MJ, Fernandes L, Paes HC,

Hahn RC, et al. Phylogenetic analysis reveals a high level of speciation in

the Paracoccidioides genus. Mol Phylogenet Evol 2009;52:273e83.



[3] McEwen JG, Bedoya V, Patino MM, Salazar ME, Restrepo A. Experi-

mental murine paracoccidiodomycosis induced by the inhalation of

conidia. J Med Vet Mycol 1987;25:165e75.

[4] Shikanai-Yasuda MA, Filho Fde Q Telles, Mendes RP, Colombo AL,

Moretti ML. Guidelines in paracoccidioidomycosis. Rev Soc Bras Med

Trop 2006;39:297e310.

[5] Prado M, Silva MB, Laurenti R, Travassos LR, Taborda CP. Mortality

due to systemic mycoses as a primary cause of death or in association

with AIDS in Brazil: a review from 1996 to 2006. Mem Inst Oswaldo

Cruz 2009;104:513e21.

[6] Silva-Vergara ML, Teixeira AC, Curi VG, Costa Jr JC, Vanunce R,

Carmo WM, et al. Paracoccidioidomycosis associated with human im-

munodeficiency virus infection. Report of 10 cases. Med Mycol

2003;41:259e63.

[7] Travassos LR, Taborda CP. New advances in the development of a

vaccine against paracoccidioidomycosis. Front Microbiol 2012;3:212.

[8] Casadevall A, Pirofski LA. Immunoglobulins in defense, pathogenesis,

and therapy of fungal diseases. Cell Host Microbe 2012;11:447e56.

[9] de Mattos Grosso D, de Almeida SR, Mariano M, Lopes JD. Charac-

terization of gp70 and anti-gp70 monoclonal antibodies in Para-

coccidioides brasiliensis pathogenesis. Infect Immun 2003;71:6534e42.

[10] Garbe TR. Heat shock proteins and infection: interactions of pathogen

and host. Experientia 1992;48:635e9.[11] Cunha DA, Zancope-Oliveira RM, Sueli M, Felipe S, Salem-Izacc SM,

Deepe Jr GS, et al. Heterologous expression, purification, and immu-

nological reactivity of a recombinant HSP60 from Paracoccidioides

brasiliensis. Clin Diagn Lab Immunol 2002;9:374e7.[12] Kaufmann SH. Heat shock proteins and the immune response. Immunol

Today 1990;11:129e36.

[13] Long KH, Gomez FJ, Morris RE, Newman SL. Identification of heat

shock protein 60 as the ligand on Histoplasma capsulatum that mediates

binding to CD18 receptors on human macrophages. J Immunol

2003;170:487e94.

[14] Guimaraes AJ, Frases S, Gomez FJ, Zancope-Oliveira RM, Nosanchuk JD.

Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of

Histoplasma capsulatum. Infect Immun 2009;77:1357e67.

[15] Izacc SM, Gomez FJ, Jesuino RS, Fonseca CA, Felipe MS, Deepe GS,

et al. Molecular cloning, characterization and expression of the heat

shock protein 60 gene from the human pathogenic fungus Para-

coccidioides brasiliensis. Med Mycol 2001;39:445e55.

[16] Salem-Izacc SM, Jesuino RS, Brito WA, Pereira M, Felipe MS,

Soares CM. Protein synthesis patterns of Paracoccidiodes brasiliensis

isolates in stage-specific forms and during cellular differentiation. J Med

Vet Mycol 1997;35:205e11.

[17] Taborda CP, Juliano MA, Puccia R, Franco M, Travassos LR. Mapping of

the T-cell epitope in the major 43-kilodalton glycoprotein of Para-

coccidioides brasiliensis which induces a Th-1 response protective against

fungal infection in BALB/c mice. Infect Immun 1998;66:786e93.

[18] Dobroff AS, Rodrigues EG, Juliano MA, Friaca DM, Nakayasu ES,

Almeida IC, et al. Differential antitumor effects of IgG and IgM

monoclonal antibodies and their synthetic complementarity-determining

regions directed to new targets of B16F10-Nex2 melanoma cells. Transl

Oncol 2010;3:204e17.[19] Kauffman CA. Histoplasmosis: a clinical and laboratory update. Clin

Microbiol Rev 2007;20:115e32.

[20] Wanke B, Aide MA. Chapter 6eparacoccidioidomycosis. J Bras Pneu-

mol 2009;35:1245e9.

[21] Laniado-Laborin R. Coccidioidomycosis and other endemic mycoses in

Mexico. Rev Iberoam Micol 2007;24:249e58.

[22] Almeida OP, Jacks Jr J, Scully C. Paracoccidioidomycosis of the mouth:

an emerging deep mycosis. Crit Rev Oral Biol Med 2003;14:377e83.

[23] de Bastos Ascenco Soares R, Gomez FJ, de Almeida Soares CM,

Deepe Jr GS. Vaccination with heat shock protein 60 induces a protective

immune response against experimental Paracoccidioides brasiliensis

pulmonary infection. Infect Immun 2008;76:4214e21.

[24] Camargo ZP, Taborda CP, Rodrigues EG, Travassos LR. The use of cell-

free antigens of Paracoccidioides brasiliensis in serological tests. J Med

Vet Mycol 1991;29:31e8.


j.micinf.2014.08.004



12345678910

11121314151617181920

[25] Buissa-Filho R, Puccia R, Marques AF, Pinto FA, Munoz JE,

Nosanchuk JD, et al. The monoclonal antibody against the major diag-

nostic antigen of Paracoccidioides brasiliensis mediates immune pro-

tection in infected BALB/c mice challenged intratracheally with the

fungus. Infect Immun 2008;76:3321e8.[26] Hahn RC, Macedo AM, Fontes CJ, Batista RD, Santos NL, Hamdan JS.

Randomly amplified polymorphic DNA as a valuable tool for epidemi-

ological studies of Paracoccidioides brasiliensis. J Clin Microbiol

2003;41:2849e54.

[27] Toledo MS, Tagliari L, Suzuki E, Silva CM, Straus AH, Takahashi HK.

Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic



fungal growth and differentiation. Characterization of monoclonal anti-

body MEST-3 directed to Manpalpha1–>3Manpalpha1–>2IPC. BMC

Microbiol 2010;10:47.

[28] Xander P, Vigna AF, Feitosa Ldos S, Pugliese L, Bailao AM, Soares CM,

et al. A surface 75-kDa protein with acid phosphatase activity recognized

by monoclonal antibodies that inhibit Paracoccidioides brasiliensis

growth. Microbes Infect 2007;9:1484e92.

[29] Hamza T, Barnett JB, Li B. Interleukin 12 a key immunoregulatory

cytokine in infection applications. Int J Mol Sci 2010;11:789e806.


/j.micinf.2014.08.004

Documents

Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental Paracoccidioides lutzii