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Microbes and Infection xx (2014) 1e8 www.elsevier.com/locate/micinf
Original article
Monoclonal antibodies to heat shock protein 60 induce a protective immune response 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,*
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a Institute of Biomedical Sciences, Department of Microbiology, Brazil Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA c Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA d 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
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b
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Received 16 September 2013; accepted 6 August 2014
Abstract Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 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 in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Paracoccidioides lutzii; Hsp60; Monoclonal antibody
1. Introduction Paracoccidioidomycosis (PCM) is caused by a complex group of fungi within the Paracoccidioides genus comprising four distinct phylogenetic lineages known as PS2, PS3, S1 and Pb01-like [1]. Pb01 is morphologically and genetically distinct from 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, Q3 Brazil. Tel.: þ55 11 3091 7351. E-mail address: [email protected] (L. Thomaz).
Paracoccidioides lutzii [2], while the others are speciated as Paracoccidioides brasiliensis. However, the pathogenesis and disease manifestations of P. brasiliensis and P. lutzii are indistinguishable at present. PCM is initiated by the inhalation of airborne conidia released by the filamentous forms of these fungi that are present in soil under certain conditions. These conidia reach the pulmonary alveoli and transition into yeast forms [3]. The disease can develop rapidly after acquisition of the conidia or manifest years after the infection. There are two main clinical forms that patients develop: acute/subacute or chronic. The acute/subacute form is more common among children and young adults, and it is characterized by
http://dx.doi.org/10.1016/j.micinf.2014.08.004 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. 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
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lymphadenopathy associated with hepatosplenomegaly as well as cutaneous, intestinal and bone lesions [4]. The chronic form typically affects adults, and it is characterized by pulmonary lesions, often associated with oral, nasal, or laryngeal ulcers. There is both an increased incidence and enhanced severity of PCM in patients co-infected with HIV [5,6]. In the absence of systemic antimicrobial therapy, PCM is uniformly progressive. Standard treatment regimens utilize sulfonamides, amphotericin B or azoles, and therapy duration is from 2 months to several years, depending on disease severity and host responsiveness [4]. Despite protracted treatment approaches, relapses are common. Regarding host responses, there is ample evidence that the activation of cellular immunity is the most effective mechanism to control experimental and human PCM, although both innate immune responses and adaptive immunity are important for manifesting effective protection (reviewed in Ref. [7]). The role of the humoral immune response in PCM is controversial especially as patients with acute and sub acute forms of the disease have high antibody titers [7]. However, for several related fungal pathogens there is considerable evidence that the administration of monoclonal antibodies (mAbs) can modify the course of experimental diseases. Protective mAbs have been successfully identified against some important fungi, such as Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum (reviewed in Refs. [7,8]). Moreover, 10 years ago a mAb to a 70 kDa glycoprotein was shown to modify experimental PCM [9]. Since this observation, several groups have begun to explore the impact of mAbs targeting Paracoccidioides complex antigens. Heat shock proteins (Hsp) have emerged as a potential target for therapeutic mAbs as they are conserved immunodominant antigens that can evoke cell mediated and humoral responses during infection [10e12]. H. capsulatum is a related dimorphic fungus that displays a 62 kDa surface form of Hsp (Hsp60) that is involved in the adhesion of the parasitic yeast form to host macrophages via interaction with CD18 integrin receptors [13]. MAbs targeting H. capsulatum Hsp60 modify the pathogenesis of murine histoplasmosis, and the protective efficacy of individual mAbs in histoplasmosis appears to correspond to the Ig subclass [14]. In the Histoplasma model, mAb 7B6, an IgG2b, increased tissue inflammation, enhanced intracellular yeast growth and was not protective, whereas IgG1 and IgG2a mAbs reduced inflammation, impeded intracellular replication, and prolonged the survival of mice [14]. Paracoccidioides spp. also expresses Hsp60 and the protein is produced in large amounts in response to rapid increases in temperature and other types of stress. The greater expression of Hsp60 in yeast cells compared to mycelium suggests that this protein could be necessary for survival in host conditions and that it may have a role in morphogenesis [15,16]. In the present study, we examined the impact of 2 mAbs generated against Hsp60 from H. capsulatum, one characterized as nonprotective (mAb 7B6, IgG2b) and another as protective (mAb 4E12, IgG2a), in a validated experimental paracoccidioidomycosis model.
2. Materials and methods 2.1. Animals BALB/c mice (6e8 weeks female) were bred at the Institute of Biomedical Sciences of University of S~ao Paulo (USP), Brazil, animal facility, under specific pathogen-free conditions. The procedures involving animals and their care were conducted according to the local ethics committee and international rules. All procedures were approved by the Ethics Committee on the Use of Animals (CEUA) of USP. 2.2. Fungal strain and growth conditions Virulent P. lutzii strain Pb01 yeast cells were grown in Sabouraud liquid medium at 37 C with shaking and were used after 5 days of growth. The fungal cells were washed in phosphate-buffered saline, pH 7.2 and counted in a hemocytometer. Cell viability was determined by staining with Trypan blue (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]. The mAbs were purified using a protein A/G affinity chromatography (Pierce, Rockland, IL) following the manufacturer's instructions. MAb concentrations were determined by Nanodrop 100 (Thermo Scientific, Wilmington, DE). 2.4. Phagocytosis assay Primary peritoneal Macrophages from BALB/c mice were cultivated in a medium containing 10% heat-inactivated fetal calf serum, 10% NCTC-109 medium (Life Technologies, Grand Island, NY), nonessential amino acids (Life Technologies) and 1% Pen-Strep. Macrophage cells were plated on 96well 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 yeast cells were collected after 5 days of growth, washed three times with PBS and incubated with or without mAb for 24 h. The mAbs were tested at two concentrations, 10 and 50 mg/ml. An irrelevant IgG (IgG2a) mAb [18] was used as an additional control. P. lutzii cells were added at 5:1 macrophage to fungal cell ratio and incubated for 12 h. The cells were then washed several times with PBS, and then with 0.15 M a-mannopyranoside (Sigma) to remove extracellular yeast. Samples were fixed with cold absolute methanol, and then stained with a 1/ 20 solution of Giemsa (Sigma). The number of phagocytosed yeast was determined by light microscopy at 400 magnification. The phagocytic index (PI) is defined as PI ¼ P F, where P is the percentage of macrophages with internalized yeast and F is the average number of yeast cells per macrophage. Experiments were carried out in triplicate. The numbers of macrophage and yeast cells were recorded for each field, and at least 200 macrophages were counted.
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
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2.5. Immunofluorescence P. lutzii yeast cells were grown as described and pelleted by centrifugation at 500 g for 5 min. The cells were washed with PBS, and 5 106 cells were suspended in 1% PBS-BSA with 100 mg/mL of mAbs anti-Hsp60, 4E12 and 7B6 or irrelevant mAb overnight at 4 C. The cells were then washed with 1% PBS-BSA three times and suspended in 1% PBSBSA with FITC-conjugated goat anti-mouse IgG secondary antibody (BD-Pharmigen) overnight at 4 C. The samples were then washed five times with 1% PBS-BSA and suspended for 10 min in 1% Calcofluor (Sigma) in PBS buffer at room temperature. The cells were then washed three times with PBS and suspended in 30 ml of N-propyl-gallate (Sigma) in glycerol (Sigma). Aliquots were placed on glass slides with cover slips and viewed by light and immunofluorescence microscopy EVOS microscope (Advanced Microscopy Group, Bothel, WA) using the AMG camera.
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with the manufacturer's instructions. The supernatants were assayed for IL-4, IL-10, IL-12 and IFN-g using ELISA kits (BD PharMingen, San Diego, CA). The detections limits of such assays were as follows: 7.8 pg/ml for IL-4; 31.25 pg/ml for 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 Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Statistical comparisons were made by analysis of variance (one-way ANOVA) followed by a Turkey-Kramer posttest. ( p) values of p < 0.05 indicated statistical significance. A 95% confidence interval was determined in all experiments. 3. Results 3.1. Immunofluorescence
2.6. Intratracheal infection of BALB/c mice BALB/c mice were each inoculated intratracheally (IT) with 50 ml of 1 106 yeast cells of Pb01. Mice were anesthetized intraperitoneally (IP) with 200 ml of a solution of 80 mg/kg ketamine/kg of body weight and 10 mg/kg of xylazine (Uni~ao Química Farmac^eutica, Brazil). Approximately 10 min after anesthetization, the neck was hyperextended, the trachea exposed at the level of thyroid, and the yeast cells were introduced via a 26-gauge needle. The incisions were sutured with 5e0 silk. 2.7. Fungal burden in organs of infected mice 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 in 1 ml of PBS. A 100 ml aliquot from each suspension was inoculated on brain-heart infusion (BHI) agar plates, supplemented with 4% fetal calf serum (Vitrocell, Brazil), 5% spent P. brasiliensis (strain 192) culture medium, streptomycin/ penicillin 10 IU/ml (Sigma). Colony countings were made after 10 days of incubation at 37 C. 2.8. Histology Sections of excised lungs were fixed in 10% buffered formalin, embedded in paraffin. The sections were stained with Grocott silver or Hematoxylin and Eosin (HE) and examined microscopically at 40 (Optiphot-2; Nikon, Tokyo, Japan). 2.9. Cytokine analysis Weighed sections of lung were homogenized in 1 ml of PBS in the presence of protease inhibitors (Sigma) to prevent degradation of cytokines by tissue proteases, centrifuged, and the supernatants frozen at e 80 C until tested in accordance
To validate the binding of mAbs 4E12 and 7B6 to P. lutzii Hsp60, we assessed the interactions of the mAbs with yeast cells by fluorescence microscopy (Fig. 1). MAb 4E12 labeled almost all P. lutzii yeast cells diffusely at the cell wall, whereas mAb 7B6 labeled yeast cells less robustly. In contrast, the irrelevant 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 macrophages from BALB/c mice. Ten and 50 mg per milliliter of either 7B6 or 4E12 significantly enhanced the phagocytosis of yeast cells compared to irrelevant mAb, p < 0.0001 (Fig. 2). 3.3. Fungal burden To evaluate whether the administration of mAbs mediated protection in BALB/c mice that received 1 mg/animal of mAbs 24 h before IT infection with P. lutzii, we examined the lungs, spleens and livers 15 days after infection. Both groups that received Hsp60-binding mAbs had significant reductions in the number of pulmonary CFUs compared with the control group treated with irrelevant mAb ( p < 0.05) (Fig. 3). However, MAb 4E12 was significantly more effective at reducing the pulmonary fungal burden compared to mAB 7B6 ( p < 0.005). No CFUs were recovered from the spleens and livers of any mice. 3.4. Lung histology Distinct differences in histology were observed between the mice that received Hsp60-binding mAbs relative to controls. In the control groups (Figs. 4 and 5A), histological review showed confluent foci of inflammation granulomas throughout the lungs and a high resolution study revealed the predominance of neutrophils as well as numerous replicating fungal
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
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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 macrophages. 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 difference 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
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Fig. 4. Gomoris methenamine silver staining reveals that Hsp60-binding mAbs reduce the number of P. lutzii in the lungs of infected mice. Gomoris 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.
cells. There were few areas of intact lung architecture in the control infected mice. In contrast, the pathological review of the lungs of mice treated with either mAbs 7B6 (Figs. 4 and 5B) or 4E12 (Figs. 4 and 5C) revealed pulmonary lesions consisting 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 treated with mAb 4E12 had lower numbers of yeast cells compared to controls or mAb 7B6 treated mice, consistent with the CFU results shown in Fig. 3, and the mice that received mAb 4E12 displayed early organization of granulomas surrounding the fungal cells (Figs. 4 and 5C). Additionally, the mAb 4E12treated mice had large areas of histologically normal lung tissue (Figs. 4 and 5). 3.5. Cytokine production Alterations in cytokine levels were detected in the lung tissues of IT infected mice treated with MAb 7B6 and 4E12 compared to mice that received irrelevant mAb (Fig. 6). The mAb 4E12 treated and infected mice had the highest levels of IFN-g ( p < 0.01) and IL-12p70 ( p < 0.05) and lowest levels of IL-4 ( p < 0.01) and IL-10 ( p < 0.01). MAb 7B6 treatment also reduced IL-4 ( p ¼ 0.049) and IL10 ( p < 0.05) levels compared to the irrelevant antibody, although to a lesser extent than those achieved with mAb 4E12. Similarly, IFN-g was increased in mAb 7B6 treated mice ( p ¼ 0.018), but IL-12p70 was not. Interestingly, mAb 7B6 significantly reduced IL12p40 levels relative to control antibody ( p < 0.05), but not in comparison with mAb 4E12 ( p > 0.05). Therefore the level
of IL-4 decreased by 37% with 7B6 and 73% with 4E12 when compared with the irrelevant antibody; for IL-10 the levels were almost 52% lower with 7B6 and 69% with 4E12; for IL12p40 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 increased 32% with 7B6 and 143% with 4E12. In conclusion, the IgG2a and 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 Latin America [7] caused by a dimorphic fungus. While histoplasmosis is the prevalent systemic fungal dimorphic mycosis in Central and North American countries [19], PCM is predominantly found in South America [20]. Approximately 80% of PCM cases in Latin America have been reported in Brazil, primarily in the states of S~ao Paulo, Parana, Rio Grande do Sul, Goias, Rio de Janeiro, and Rond^onia [21,22]. PCM, histoplasmosis, coccidioidomycosis, and blastomycosis share some important attributes, including dimorphism, immunodominant antigens and several clinical and pathological characteristics [23]. Although antifungal therapy is the universally used treatment of PCM, the passive transfer of specific mAb may boost cellular immune responses and augment the protective effect of chemotherapy [7]. In 2003, two IgG1 mAbs to P. brasiliensis 70 kDa glycoprotein, which is recognized by 96% sera from PCM patients, were highly protective in experimental murine PCM when
Fig. 5. Hsp60-binding mAbs reduce the number of P. lutzii in the lungs of infected mice. Hematoxylin and Eosin 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. 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
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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.
administered at the time of infection [9]. The histology of lung sections from mice infected with P. brasiliensis and treated with mAb revealed significantly reduced fungal burdens and a protective cellular response characterized by granuloma formation. The main diagnostic antigen for P. brasiliensis, the gp43, which is recognized by virtually 100% of patients with PCM [24], has also been explored as a target for antibody therapy. Previous data from our group demonstrated that the passive administration of mAb 3E (IgG2b) to gp43 before and after intratracheal infection or intravenous infection reduced fungal burden and decreased pulmonary infection, whereas mAb 32H (IgG2a) was not protective [25]. We also investigated the effect of mAbs to gp43 on phagocytosis using primary macrophages (lung and peritoneal) and cell lines (J774.16 and MH-S). Yeast cells opsonized with protective mAbs were internalized at least twofold more than yeast cells in the absence of antibody or opsonized by non-protective mAbs. The results with primary macrophages and cell lines were similar [25].
In addition to belonging to different isotypes, these gp43binding mAbs react with distinct epitopes. It should be noted that patients infected with P. lutzii usually have negative serological responses in gp43-antigen based assays [26]. An IgG2a mAb to P. brasiliensis glycosphingolipids reduced the growth and morphogenesis of P. brasiliensis, H. capsulatum, and Sporothrix schenckii, in vitro [27]. IgM and IgG2a mAbs to gp75, a P. brasiliensis protein with phosphatase activity, inhibited fungal growth in vitro and passive immunization using either mAb prior to and during infection reduced pulmonary fungal burdens and the lungs of the treated mice were histologically normal after 45 days infection [28]. Given the high level of conservation among the dimorphic fungal Hsp family, and particularly between H. capsulatum and the P. brasiliensis Hsp60 complex [23], we tested the effects of mAbs generated to H. capsulatum Hsp60 in an experimental P. lutzii infection model. In murine histoplasmosis, the IgG2a mAb prolonged the survival of mice associated to a strong Th-1 response while the IgG2b mAb
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
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exacerbated disease and polarized the host reaction toward a Th-2 response [14]. In the present work, we showed that mAbs 4E12 and 7B6 were biologically active against P. lutzii. Both isotypes enhanced P. lutzii phagocytosis in vitro. Passive administration of the mAbs prior to IT infection of P. lutzii in mice significantly reduced the fungal burden in pulmonary tissue. This contrasts with the disease enhancing effect of mAb 7B6 in histoplasmosis. Moreover, as assessed by histology, both mAbs reduced the pulmonary tissue damage due to PCM, albeit the protective effect of mAb 4E12 was significantly more pronounced. This may be attributed to the more robust binding, as determined by fluorescence assays, of mAb 4E12 to P. lutzii yeast cells relative to that observed with mAb 7B6. Additionally, the formation of compact granulomas in the mice treated with mAb 4E12 can be associated with the robust Th-1 biased response identified by the cytokine assays. Although mAb 7B6 was associated with a Th-2 response in histoplasmosis, we found that this mAb contributed to a Th-1 biased response in PCM. Interestingly, mice treated with mAb 7B6 had significantly lower levels of IL-12p40 than control animals. IL-12p40 may block IL-12p70 associated responses [29], so that a reduction in IL-12p40 may be a protective response even in face of a lower induction of IL-12p70 by mAb 7B6 relative to MAb 4E12. The role of IFN-gamma mediating activated macrophages in paracoccidioidomycosis has been well documented [reviewed in 25]. Murine peritoneal macrophages and cell line activated by IFN-gamma show enhanced fungicidal activity against P. brasiliensis [25]. However, we showed here a similar mechanism against P. lutzii in which mAbs 7B6 and 4E12 significantly enhance IFNgamma production. In conclusion, we demonstrate the protective efficacy of mAbs raised to H. capsulatum Hsp60 in a murine PCM model. Moreover, we have found significant differences in the infection outcomes as MAb 7B6 exacerbates histoplasmosis while it is protective in PCM. This striking observation suggests that key differences may happen in the biology of these fungal pathogens or in the manner in which mammalian hosts respond to their presence. Future studies will be undertaken to explore the mechanisms behind these differences. Q4
Acknowledgments The present work was supported by FAPESP grant 2007/ 53175-1, 2011/17267-4, 2013/18655-3. CPT and LRT are career 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 Paracoccidioides 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.
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[3] McEwen JG, Bedoya V, Patino MM, Salazar ME, Restrepo A. Experimental 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 immunodeficiency 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. Characterization of gp70 and anti-gp70 monoclonal antibodies in Paracoccidioides 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 immunological 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 Paracoccidioides 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 Paracoccidioides 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 Pneumol 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 cellfree antigens of Paracoccidioides brasiliensis in serological tests. J Med Vet Mycol 1991;29:31e8.
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[25] Buissa-Filho R, Puccia R, Marques AF, Pinto FA, Munoz JE, Nosanchuk JD, et al. The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus. Infect Immun 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 epidemiological 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 antibody 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.
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Microbes and Infection xx (2014) 1e8 www.elsevier.com/locate/micinf
Original article
Monoclonal antibodies to heat shock protein 60 induce a protective immune response 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,*
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a Institute of Biomedical Sciences, Department of Microbiology, Brazil Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA c Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA d 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
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b
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Received 16 September 2013; accepted 6 August 2014
Abstract Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 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 in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Paracoccidioides lutzii; Hsp60; Monoclonal antibody
1. Introduction Paracoccidioidomycosis (PCM) is caused by a complex group of fungi within the Paracoccidioides genus comprising four distinct phylogenetic lineages known as PS2, PS3, S1 and Pb01-like [1]. Pb01 is morphologically and genetically distinct from 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, Q3 Brazil. Tel.: þ55 11 3091 7351. E-mail address: [email protected] (L. Thomaz).
Paracoccidioides lutzii [2], while the others are speciated as Paracoccidioides brasiliensis. However, the pathogenesis and disease manifestations of P. brasiliensis and P. lutzii are indistinguishable at present. PCM is initiated by the inhalation of airborne conidia released by the filamentous forms of these fungi that are present in soil under certain conditions. These conidia reach the pulmonary alveoli and transition into yeast forms [3]. The disease can develop rapidly after acquisition of the conidia or manifest years after the infection. There are two main clinical forms that patients develop: acute/subacute or chronic. The acute/subacute form is more common among children and young adults, and it is characterized by
http://dx.doi.org/10.1016/j.micinf.2014.08.004 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. 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
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lymphadenopathy associated with hepatosplenomegaly as well as cutaneous, intestinal and bone lesions [4]. The chronic form typically affects adults, and it is characterized by pulmonary lesions, often associated with oral, nasal, or laryngeal ulcers. There is both an increased incidence and enhanced severity of PCM in patients co-infected with HIV [5,6]. In the absence of systemic antimicrobial therapy, PCM is uniformly progressive. Standard treatment regimens utilize sulfonamides, amphotericin B or azoles, and therapy duration is from 2 months to several years, depending on disease severity and host responsiveness [4]. Despite protracted treatment approaches, relapses are common. Regarding host responses, there is ample evidence that the activation of cellular immunity is the most effective mechanism to control experimental and human PCM, although both innate immune responses and adaptive immunity are important for manifesting effective protection (reviewed in Ref. [7]). The role of the humoral immune response in PCM is controversial especially as patients with acute and sub acute forms of the disease have high antibody titers [7]. However, for several related fungal pathogens there is considerable evidence that the administration of monoclonal antibodies (mAbs) can modify the course of experimental diseases. Protective mAbs have been successfully identified against some important fungi, such as Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum (reviewed in Refs. [7,8]). Moreover, 10 years ago a mAb to a 70 kDa glycoprotein was shown to modify experimental PCM [9]. Since this observation, several groups have begun to explore the impact of mAbs targeting Paracoccidioides complex antigens. Heat shock proteins (Hsp) have emerged as a potential target for therapeutic mAbs as they are conserved immunodominant antigens that can evoke cell mediated and humoral responses during infection [10e12]. H. capsulatum is a related dimorphic fungus that displays a 62 kDa surface form of Hsp (Hsp60) that is involved in the adhesion of the parasitic yeast form to host macrophages via interaction with CD18 integrin receptors [13]. MAbs targeting H. capsulatum Hsp60 modify the pathogenesis of murine histoplasmosis, and the protective efficacy of individual mAbs in histoplasmosis appears to correspond to the Ig subclass [14]. In the Histoplasma model, mAb 7B6, an IgG2b, increased tissue inflammation, enhanced intracellular yeast growth and was not protective, whereas IgG1 and IgG2a mAbs reduced inflammation, impeded intracellular replication, and prolonged the survival of mice [14]. Paracoccidioides spp. also expresses Hsp60 and the protein is produced in large amounts in response to rapid increases in temperature and other types of stress. The greater expression of Hsp60 in yeast cells compared to mycelium suggests that this protein could be necessary for survival in host conditions and that it may have a role in morphogenesis [15,16]. In the present study, we examined the impact of 2 mAbs generated against Hsp60 from H. capsulatum, one characterized as nonprotective (mAb 7B6, IgG2b) and another as protective (mAb 4E12, IgG2a), in a validated experimental paracoccidioidomycosis model.
2. Materials and methods 2.1. Animals BALB/c mice (6e8 weeks female) were bred at the Institute of Biomedical Sciences of University of S~ao Paulo (USP), Brazil, animal facility, under specific pathogen-free conditions. The procedures involving animals and their care were conducted according to the local ethics committee and international rules. All procedures were approved by the Ethics Committee on the Use of Animals (CEUA) of USP. 2.2. Fungal strain and growth conditions Virulent P. lutzii strain Pb01 yeast cells were grown in Sabouraud liquid medium at 37 C with shaking and were used after 5 days of growth. The fungal cells were washed in phosphate-buffered saline, pH 7.2 and counted in a hemocytometer. Cell viability was determined by staining with Trypan blue (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]. The mAbs were purified using a protein A/G affinity chromatography (Pierce, Rockland, IL) following the manufacturer's instructions. MAb concentrations were determined by Nanodrop 100 (Thermo Scientific, Wilmington, DE). 2.4. Phagocytosis assay Primary peritoneal Macrophages from BALB/c mice were cultivated in a medium containing 10% heat-inactivated fetal calf serum, 10% NCTC-109 medium (Life Technologies, Grand Island, NY), nonessential amino acids (Life Technologies) and 1% Pen-Strep. Macrophage cells were plated on 96well 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 yeast cells were collected after 5 days of growth, washed three times with PBS and incubated with or without mAb for 24 h. The mAbs were tested at two concentrations, 10 and 50 mg/ml. An irrelevant IgG (IgG2a) mAb [18] was used as an additional control. P. lutzii cells were added at 5:1 macrophage to fungal cell ratio and incubated for 12 h. The cells were then washed several times with PBS, and then with 0.15 M a-mannopyranoside (Sigma) to remove extracellular yeast. Samples were fixed with cold absolute methanol, and then stained with a 1/ 20 solution of Giemsa (Sigma). The number of phagocytosed yeast was determined by light microscopy at 400 magnification. The phagocytic index (PI) is defined as PI ¼ P F, where P is the percentage of macrophages with internalized yeast and F is the average number of yeast cells per macrophage. Experiments were carried out in triplicate. The numbers of macrophage and yeast cells were recorded for each field, and at least 200 macrophages were counted.
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2.5. Immunofluorescence P. lutzii yeast cells were grown as described and pelleted by centrifugation at 500 g for 5 min. The cells were washed with PBS, and 5 106 cells were suspended in 1% PBS-BSA with 100 mg/mL of mAbs anti-Hsp60, 4E12 and 7B6 or irrelevant mAb overnight at 4 C. The cells were then washed with 1% PBS-BSA three times and suspended in 1% PBSBSA with FITC-conjugated goat anti-mouse IgG secondary antibody (BD-Pharmigen) overnight at 4 C. The samples were then washed five times with 1% PBS-BSA and suspended for 10 min in 1% Calcofluor (Sigma) in PBS buffer at room temperature. The cells were then washed three times with PBS and suspended in 30 ml of N-propyl-gallate (Sigma) in glycerol (Sigma). Aliquots were placed on glass slides with cover slips and viewed by light and immunofluorescence microscopy EVOS microscope (Advanced Microscopy Group, Bothel, WA) using the AMG camera.
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with the manufacturer's instructions. The supernatants were assayed for IL-4, IL-10, IL-12 and IFN-g using ELISA kits (BD PharMingen, San Diego, CA). The detections limits of such assays were as follows: 7.8 pg/ml for IL-4; 31.25 pg/ml for 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 Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Statistical comparisons were made by analysis of variance (one-way ANOVA) followed by a Turkey-Kramer posttest. ( p) values of p < 0.05 indicated statistical significance. A 95% confidence interval was determined in all experiments. 3. Results 3.1. Immunofluorescence
2.6. Intratracheal infection of BALB/c mice BALB/c mice were each inoculated intratracheally (IT) with 50 ml of 1 106 yeast cells of Pb01. Mice were anesthetized intraperitoneally (IP) with 200 ml of a solution of 80 mg/kg ketamine/kg of body weight and 10 mg/kg of xylazine (Uni~ao Química Farmac^eutica, Brazil). Approximately 10 min after anesthetization, the neck was hyperextended, the trachea exposed at the level of thyroid, and the yeast cells were introduced via a 26-gauge needle. The incisions were sutured with 5e0 silk. 2.7. Fungal burden in organs of infected mice 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 in 1 ml of PBS. A 100 ml aliquot from each suspension was inoculated on brain-heart infusion (BHI) agar plates, supplemented with 4% fetal calf serum (Vitrocell, Brazil), 5% spent P. brasiliensis (strain 192) culture medium, streptomycin/ penicillin 10 IU/ml (Sigma). Colony countings were made after 10 days of incubation at 37 C. 2.8. Histology Sections of excised lungs were fixed in 10% buffered formalin, embedded in paraffin. The sections were stained with Grocott silver or Hematoxylin and Eosin (HE) and examined microscopically at 40 (Optiphot-2; Nikon, Tokyo, Japan). 2.9. Cytokine analysis Weighed sections of lung were homogenized in 1 ml of PBS in the presence of protease inhibitors (Sigma) to prevent degradation of cytokines by tissue proteases, centrifuged, and the supernatants frozen at e 80 C until tested in accordance
To validate the binding of mAbs 4E12 and 7B6 to P. lutzii Hsp60, we assessed the interactions of the mAbs with yeast cells by fluorescence microscopy (Fig. 1). MAb 4E12 labeled almost all P. lutzii yeast cells diffusely at the cell wall, whereas mAb 7B6 labeled yeast cells less robustly. In contrast, the irrelevant 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 macrophages from BALB/c mice. Ten and 50 mg per milliliter of either 7B6 or 4E12 significantly enhanced the phagocytosis of yeast cells compared to irrelevant mAb, p < 0.0001 (Fig. 2). 3.3. Fungal burden To evaluate whether the administration of mAbs mediated protection in BALB/c mice that received 1 mg/animal of mAbs 24 h before IT infection with P. lutzii, we examined the lungs, spleens and livers 15 days after infection. Both groups that received Hsp60-binding mAbs had significant reductions in the number of pulmonary CFUs compared with the control group treated with irrelevant mAb ( p < 0.05) (Fig. 3). However, MAb 4E12 was significantly more effective at reducing the pulmonary fungal burden compared to mAB 7B6 ( p < 0.005). No CFUs were recovered from the spleens and livers of any mice. 3.4. Lung histology Distinct differences in histology were observed between the mice that received Hsp60-binding mAbs relative to controls. In the control groups (Figs. 4 and 5A), histological review showed confluent foci of inflammation granulomas throughout the lungs and a high resolution study revealed the predominance of neutrophils as well as numerous replicating fungal
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
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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 macrophages. 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 difference relative to the irrelevant control.
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Fig. 4. Gomoris methenamine silver staining reveals that Hsp60-binding mAbs reduce the number of P. lutzii in the lungs of infected mice. Gomoris 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.
cells. There were few areas of intact lung architecture in the control infected mice. In contrast, the pathological review of the lungs of mice treated with either mAbs 7B6 (Figs. 4 and 5B) or 4E12 (Figs. 4 and 5C) revealed pulmonary lesions consisting 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 treated with mAb 4E12 had lower numbers of yeast cells compared to controls or mAb 7B6 treated mice, consistent with the CFU results shown in Fig. 3, and the mice that received mAb 4E12 displayed early organization of granulomas surrounding the fungal cells (Figs. 4 and 5C). Additionally, the mAb 4E12treated mice had large areas of histologically normal lung tissue (Figs. 4 and 5). 3.5. Cytokine production Alterations in cytokine levels were detected in the lung tissues of IT infected mice treated with MAb 7B6 and 4E12 compared to mice that received irrelevant mAb (Fig. 6). The mAb 4E12 treated and infected mice had the highest levels of IFN-g ( p < 0.01) and IL-12p70 ( p < 0.05) and lowest levels of IL-4 ( p < 0.01) and IL-10 ( p < 0.01). MAb 7B6 treatment also reduced IL-4 ( p ¼ 0.049) and IL10 ( p < 0.05) levels compared to the irrelevant antibody, although to a lesser extent than those achieved with mAb 4E12. Similarly, IFN-g was increased in mAb 7B6 treated mice ( p ¼ 0.018), but IL-12p70 was not. Interestingly, mAb 7B6 significantly reduced IL12p40 levels relative to control antibody ( p < 0.05), but not in comparison with mAb 4E12 ( p > 0.05). Therefore the level
of IL-4 decreased by 37% with 7B6 and 73% with 4E12 when compared with the irrelevant antibody; for IL-10 the levels were almost 52% lower with 7B6 and 69% with 4E12; for IL12p40 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 increased 32% with 7B6 and 143% with 4E12. In conclusion, the IgG2a and 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 Latin America [7] caused by a dimorphic fungus. While histoplasmosis is the prevalent systemic fungal dimorphic mycosis in Central and North American countries [19], PCM is predominantly found in South America [20]. Approximately 80% of PCM cases in Latin America have been reported in Brazil, primarily in the states of S~ao Paulo, Parana, Rio Grande do Sul, Goias, Rio de Janeiro, and Rond^onia [21,22]. PCM, histoplasmosis, coccidioidomycosis, and blastomycosis share some important attributes, including dimorphism, immunodominant antigens and several clinical and pathological characteristics [23]. Although antifungal therapy is the universally used treatment of PCM, the passive transfer of specific mAb may boost cellular immune responses and augment the protective effect of chemotherapy [7]. In 2003, two IgG1 mAbs to P. brasiliensis 70 kDa glycoprotein, which is recognized by 96% sera from PCM patients, were highly protective in experimental murine PCM when
Fig. 5. Hsp60-binding mAbs reduce the number of P. lutzii in the lungs of infected mice. Hematoxylin and Eosin 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. 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
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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.
administered at the time of infection [9]. The histology of lung sections from mice infected with P. brasiliensis and treated with mAb revealed significantly reduced fungal burdens and a protective cellular response characterized by granuloma formation. The main diagnostic antigen for P. brasiliensis, the gp43, which is recognized by virtually 100% of patients with PCM [24], has also been explored as a target for antibody therapy. Previous data from our group demonstrated that the passive administration of mAb 3E (IgG2b) to gp43 before and after intratracheal infection or intravenous infection reduced fungal burden and decreased pulmonary infection, whereas mAb 32H (IgG2a) was not protective [25]. We also investigated the effect of mAbs to gp43 on phagocytosis using primary macrophages (lung and peritoneal) and cell lines (J774.16 and MH-S). Yeast cells opsonized with protective mAbs were internalized at least twofold more than yeast cells in the absence of antibody or opsonized by non-protective mAbs. The results with primary macrophages and cell lines were similar [25].
In addition to belonging to different isotypes, these gp43binding mAbs react with distinct epitopes. It should be noted that patients infected with P. lutzii usually have negative serological responses in gp43-antigen based assays [26]. An IgG2a mAb to P. brasiliensis glycosphingolipids reduced the growth and morphogenesis of P. brasiliensis, H. capsulatum, and Sporothrix schenckii, in vitro [27]. IgM and IgG2a mAbs to gp75, a P. brasiliensis protein with phosphatase activity, inhibited fungal growth in vitro and passive immunization using either mAb prior to and during infection reduced pulmonary fungal burdens and the lungs of the treated mice were histologically normal after 45 days infection [28]. Given the high level of conservation among the dimorphic fungal Hsp family, and particularly between H. capsulatum and the P. brasiliensis Hsp60 complex [23], we tested the effects of mAbs generated to H. capsulatum Hsp60 in an experimental P. lutzii infection model. In murine histoplasmosis, the IgG2a mAb prolonged the survival of mice associated to a strong Th-1 response while the IgG2b mAb
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
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exacerbated disease and polarized the host reaction toward a Th-2 response [14]. In the present work, we showed that mAbs 4E12 and 7B6 were biologically active against P. lutzii. Both isotypes enhanced P. lutzii phagocytosis in vitro. Passive administration of the mAbs prior to IT infection of P. lutzii in mice significantly reduced the fungal burden in pulmonary tissue. This contrasts with the disease enhancing effect of mAb 7B6 in histoplasmosis. Moreover, as assessed by histology, both mAbs reduced the pulmonary tissue damage due to PCM, albeit the protective effect of mAb 4E12 was significantly more pronounced. This may be attributed to the more robust binding, as determined by fluorescence assays, of mAb 4E12 to P. lutzii yeast cells relative to that observed with mAb 7B6. Additionally, the formation of compact granulomas in the mice treated with mAb 4E12 can be associated with the robust Th-1 biased response identified by the cytokine assays. Although mAb 7B6 was associated with a Th-2 response in histoplasmosis, we found that this mAb contributed to a Th-1 biased response in PCM. Interestingly, mice treated with mAb 7B6 had significantly lower levels of IL-12p40 than control animals. IL-12p40 may block IL-12p70 associated responses [29], so that a reduction in IL-12p40 may be a protective response even in face of a lower induction of IL-12p70 by mAb 7B6 relative to MAb 4E12. The role of IFN-gamma mediating activated macrophages in paracoccidioidomycosis has been well documented [reviewed in 25]. Murine peritoneal macrophages and cell line activated by IFN-gamma show enhanced fungicidal activity against P. brasiliensis [25]. However, we showed here a similar mechanism against P. lutzii in which mAbs 7B6 and 4E12 significantly enhance IFNgamma production. In conclusion, we demonstrate the protective efficacy of mAbs raised to H. capsulatum Hsp60 in a murine PCM model. Moreover, we have found significant differences in the infection outcomes as MAb 7B6 exacerbates histoplasmosis while it is protective in PCM. This striking observation suggests that key differences may happen in the biology of these fungal pathogens or in the manner in which mammalian hosts respond to their presence. Future studies will be undertaken to explore the mechanisms behind these differences. Q4
Acknowledgments The present work was supported by FAPESP grant 2007/ 53175-1, 2011/17267-4, 2013/18655-3. CPT and LRT are career 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 Paracoccidioides 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.
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