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. 2004 Jul;137(1):123–128. doi: 10.1111/j.1365-2249.2004.02507.x

B and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2β) mimicking gp43 from Paracoccidioides brasiliensis

E B SOUZA †, J D LOPES *, S R ALMEIDA †
PMCID: PMC1809080  PMID: 15196252

Abstract

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 kD (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM). Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3). To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria. To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested. Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2. However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43. Our results also showed that, when mice were immunized with Ab2β and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.

Keywords: Paracoccidioides brasiliensis, gp43, idiotypic network, monoclonal antibodies

INTRODUCTION

Paracoccidioidomycosis (PCM) is a deep mycosis characterized by suppurative granulomatous inflammation, suppression of cellular immunity and high antibody titres [1,2]. The disease may develop as multiple forms, ranging from benign and localized to severe and disseminated forms depending upon the extent of depression of cellular immunity [2–4]. The aetiological agent of this mycosis is Paracoccidioides brasiliensis (P.b), a thermally dimorphic fungus. Airborne fungal propaguli start the infection with micelial forms that in the lungs undergo conversion to the yeast phase, the infective form of P.b [4,5].

Most exposed subjects develop an asymptomatic infection (PCM-infection) with well-organized granulomatous lesions in the lungs and lymphnodes called primary complexes. In some individuals these lesions are rich in viable fungi, can remain in latent stage and after some years disseminate to other. organs and tissues. When such dissemination happens quickly, the form is called acute [4–6].

The benign form presents preserved cellular immunity and moderate specific antibody response, while high levels of humoral immune response and depression of cell-mediated immunity are typical of the disseminated infection [7,8]. The host's defensive capacity is the most important mechanisms against PCM-infection. Thus, a correlation between the clinical forms and the pattern of the immune response development has been investigated both, in man and in the mouse model.

The main antigenic component of P. brasiliensis is a 43-kD surface glycoprotein (gp43). Gp43 is biochemically characterized as a high-mannose concanavalin A (Con A) binding glycoprotein [9]. Since gp43 is recognized by all patients' sera, the molecule is used in serological assays for diagnostic purposes [10,11]. Other biological functions have been proposed for this glycoprotein such as a laminin-binding protein implicated in fungal pathogenesis in vivo, and its expressed immunodominant epitopes elicit T cell-dependent delayed hypersensitivity reaction, thus inducing a T CD4 + lymphocyte proliferation response in humans and experimental animals.

The role played by anti-gp43 antibodies in the evolution and maintenance of PCM infection is not fully understood. Antibodies against this glycoprotein can be detected during all stages of the disease but most authors consider them as nonprotective and unrelated to fungal pathogenesis.

According to the network hypothesis proposed by Jerne [12], anti-idiotypic (anti-Id) antibodies are part of the normal immune response, resulting in a web of interacting idiotypic (Id) antibodies. Idiotypes (Id) are the sum of idiotopes or serologically determined antigenic determinants, unique to an antibody or group of antibodies. The potential immune regulatory role of Id–anti-Id interactions has been intensively investigated [13,14]. Anti-Id antibodies recognizing antigenic determinants that overlap a portion of the combining site that is in contact with the original antigen are said to carry its ‘internal image’. Although those anti-Id antibodies, also known as Ab2β, are able to mimic the antigen, they represent a small fraction of all anti-Id antibodies produced [15]. By definition, Ab2β compete with the external antigen in the binding to antibodies (Ab1) raised against the latter [16]. This mimicry points to the relevance of these antibodies from immunological and biological points of view. Also, the therapeutic potential of anti-Id antibodies has been exploited in different systems [14,15,17].

Recently, Souza et al. [18] have shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1) unleashes the idiotypic cascade, producing both anti-Id antibodies (Ab2) as well as anti-anti-Id antibodies (Ab3). Moreover, it was found for the first time that PCM patient's sera also displayed significant amounts of Ab2. These results pointed to spontaneous modulation of the idiotypic cascade in the gp43 system of P. brasiliensis in both, mice and humans. To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas simultaneously producing MAbs Ab2 and Ab3 were obtained from fused spleen cells of the same animal. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb binds to the idiotope, thus fulfilling the ‘internal image’ criteria proposed by Nisonoff & Lamoyi [16].

Since Ab2β 7.B12 mimics of the antigen epitope of gp43 from P. brasiliensis, in this work we investigated whether this antibody could act as antigen surrogate in serological assays and could induce an antigen-specific T cell immunity.

MATERIALS AND METHODS

Animals

Six-week-old female BALB/c mice were obtained from the São Paulo University animal facilities.

Human serum specimens

Individual serum specimens from 25 patients with PCM, 5 from patients with Acute Form (AF) and 20 from patients with Chronic Form (CF), were selected based on clinical diagnosis of the disease and confirmed by positive direct examination of chraracteristic multiple budding yeast forms either in histopathologic sections or in biological fluids. All patients sera were found positive for anti-gp43 antibodies by both, immunodiffusion test and capture enzyme immunoassay (EIA), which was shown to be more sensitive and specific. These sera were collected before anti-mycotic therapy. Five patients were given anti-mycotic therapy (three with itraconazole, one with fluconazole, and one with a sulfamide derivative), and one was considered cured along the time of this study. Serum specimens from patients with Histoplasmosis, Cripotococcosis, Candidiasis and Aspergilosis were selected based on clinical diagnosis of the disease and confirmed by positive direct examination either in histopathologic sections or in biological fluids. All serum specimens, from healthy individuals or fungal infections, were obtained from Hospital São Paulo, Federal University of São Paulo.

Preparation of fungal antigens

For gp43 purification, exoantigen of P. brasiliensis B-339 was prepared as previously described [11] and passed through an adsorbent column consisting of murine anti-gp43 monoclonal antibody (MAb) 17c (IgG2a, κ light chain) [19] coupled to Affi-gel 10 column (Bio-Rad Laboratories, Hercules, CA, USA). Gp43 was eluted with 0·1 m citric acid buffer (pH 2·8), neutralized with 1 m Tris (pH 9·0), and further concentrated in a 10K Amicon apparatus (Amicon Division, Beverly, MA, USA). Deglycosylation of affinity-purified gp43 was performed with recombinant PGNase (New England Beverly, MA, USA) as described elsewhere [19]. Protein contents were determined by the Bradford method [20] and every gp43 purification steps were monitored by sodium dodecyl sulphate-polyacrylamide gel eletrophoresis (SDS-PAGE) [21].

Obtaining of MAb Ab2β 7.B12

Hybridoma was produced as described previously [18]. Large amounts of monoclonal antibodies 7B12 (IgG1, κ light chain) were obtained by production of ascites in BALB/c mice previously primed with Pristane (Sigma). MAbs were purified from ascites fluids by affinity chromatography in a Protein G column. The antibodies were dialysed against PBS and quantified.

Detection of anti-gp43 antibodies in human sera using anti-Id MAb as antigen surrogate

Anti-Id MAb 7.B12 (50, 100 or 200 µg/ml) was used to coat polyvinyl microplates and free sites were blocked with PBS containing 1% bovine serum albumin (PBS-BSA 1%) for 2 h at 37°C. Wells were treated with PBS-diluted human PCM sera for 1 h at 37°C. After washing with PBS containing 0·5% gelatin (Difco Laboratories, Detroit, MI, USA) and 0·05% Tween 20 (Sigma Chemical Co., St Louis, MO, USA) (PBS-T-G), microplates were treated with peroxidase-labelled goat anti-human IgG, washed exhaustively with PBS-T-G. Reactions were developed with o-phenylenediamine (OPD) in 0·1 m acetate-phosphate buffer pH 5·8, interrupted with 4 N H2SO4 and read in a Titertek Multiscan EIA reader at 492 nm. Heterologous sera were used in the same conditions described above for PCM sera's [22]. As control, another plate was coated with irrelevant monoclonal antibody (MAb anticarcinoembryonic antigen-CEA 1.H2, IgG1, κ light chain) [18], in the same condition described above. After preliminary experiments, the final concentration of Ab2 used in all experiments was determined as 50 µg/ml and dilution sera at 1 : 100.

Immunization of mice

MAb Ab2 7.B12 was emulsified in complete Freund's adjuvant (Sigma Chemical Co.). 50 µl of the emulsion containing 50 µg of the antibody was subcutaneously injected in the hind footpads and in the tail base of mice. One week later, the animals were sacrificed and inguinal, para-aorthic and popliteal lymph nodes were excised for cell isolation. As control, mice were immunized with 50 µg of irrelevant monoclonal antibody 1.H2 emulsified in complete Freund's adjuvant.

Proliferative response assays

Lymphnode cell suspensions in complete medium were plated, at a concentration of 2 × 105 cells per well, to 96-well plates in the presence of gp43 (5 and10 µg/ml), Ab2 (2, 3, 4 and 5 µg/ml) or irrelevant MAb 1.H2 (2, 3, 4 and 5 µg/ml). Cells were cocultured for 72 h and then pulsed for 16 h with [3H]-labelled Thymidine (1 µCi/ml). Cells were collected with an automated cell harvester, and incorporated radioactivity was measured by liquid scintillation spectrometry. Data are expressed as mean ± standard deviation counts per minute of [3H]-thymidine incorporation.

Cytokine assays

Interferon-γ (IFN-γ), IL-2, IL-4 and IL-10 from lymphonode cultures were quantified by enzyme-linked immunoabsorbent assay (Pharmingen, San Diego, CA, USA), according to the manufacturer's instructions. Briefly, cell supernatants were obtained by culturing 4 × 106 cells/ml in 24-well plates (Costar), in the presence or absence of gp43 (5 µg/ml), Ab2 (4 µg/ml) or irrelevant MAb 1.H2 (4 µg/ml), in 1 ml final volume of complete medium. After 72 h, the supernatants were colleted and interleukin quantities determined. For this propose, were employed curves with serial dilutions of mouse recombinant IFN-γ, IL-2, IL-4 and IL-10. Results were expressed as the mean ± standard deviation obtained from three experiments.

RESULTS

Detection of anti-gp43 antibodies sera of different clinical forms of PCM

Figure 1 shows the anti-gp43 titres in sera of PCM patients with different clinical forms (all diluted 1 : 100) by ELISA assays using plates coated with 50 µg/ml of Ab2. Patients with acute form showed higher titres of anti-gp43 that bind to Ab2 when compared with chronic form.

Fig. 1.

Fig. 1

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Detection of anti-gp43 antibodies in sera of patients with different clinical forms of PCM, using anti- Id MAb 7.B12 as gp43 surrogate. Sera of healthy individuals were used as negative controls. Each datum point represents the results for each patient. O.D., optical density. Means for anti-gp43 antibodies are indicated (horizontal lines).

Reaction of Ab2 with heterologous sera

In order to investigate whether antibodies from patients with other fungal infections could react with Ab2, an ELISA assay was realized. It was observed that all heterologous sera reacted with Ab2, despite presenting lower titres than PCM patients (Fig. 2). To analyse the cross-reactions, plate was coated with irrelevant MAb. The results showed that both, heterologous and PCM sera reacted with irrelevant MAb (Fig. 3), suggesting that cross-reactions observed in Fig. 2 were not related with gp43 binding epitope. In added, no inhibition in ELISA assay using MAb 7B12 coating plate was observed when heterologous sera were previously incubated with deglycosylated form of gp43 (p38) (data not show).

Fig. 2.

Fig. 2

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Results for the different clinical groups for anti-gp43 by ELISA using anti-Id MAb 7B12 as gp43 surrogate. PCM, paracoccidioidomycosis; Hist, Histoplasmosis; Crip, Criptococosis; Cand, Candidiasis; Asper, Aspergillosis.

Fig. 3.

Fig. 3

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Unspecific binding of different clinical groups using plate coated with irrelevant MAb 1.H2. Plate was coated with 50 µg/ml of irrelevant MAb and serum was used at 1 : 100. PCM, paracoccidioidomycosis; Hist, Histoplasmosis; Crip, Criptococosis; Cand, Candidiasis; Asper, Aspergillosis.

Follow-up of binding anti-gp43 antibodies to Ab2 in patients given antimycotic therapy

Some PCM patients were serologically monitored along the course of treatment and the usefulness of the ELISA assay with microplate coated with Ab2 was assessed. Figure 4 shows anti-gp43 titres in the sera from five patients with acute form of disease after 0, 6 and 12 months of antimycotic therapy using plates coated with either Ab2 or gp43. Results showed that anti-gp43 titres decayed along the treatment for both coatings used.

Fig. 4.

Fig. 4

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Follow-up of anti-gp43 antibody titres in patients given antimycotic therapy using plates coated with anti-Id MAb 7.B12 (▪) or purified gp43 (▴). Sera of patients with AF of PCM were assayed for the presence of anti-gp43 antibodies at 0, 6 and 12 months after antimycotic treatment. Datum points represent optical density (O.D) values. Plate was coated with 50 µg/ml of 7B12 Mab and serum was used at 1 : 100. Error bars indicate standard deviations.

Proliferation and cytokine production of MAb Ab2 primed T cells

To investigate induction of cellular response by Ab2, proliferation response of T cells from immunized mice was realized. Interestingly, Ab2 sensitized T cells, when re-stimulated in vitro, lead to proliferation in the presence of Ab2, but did so also in the presence of gp43. No cross-reaction was observed when immune cells were re-stimulated with irrelevant MAb (Fig. 5). Also, no cross-reaction was observed when mice were immunized with irrelevant MAb and re-stimulated with gp43 or Ab2 (Fig. 6). When cytokine patterns were evaluated, it was seen that stimulation by gp43 in vitro of Ab2 primed T cells produced high levels of IFN-γ (Fig. 7) and no secretion of IL-2, IL-4 and IL-10 (data not shown). Also, no cross-reaction was observed when Ab2 primed T cells were re-stimulated in vitro with irrelevant MAb.

Fig. 5.

Fig. 5

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Proliferation of immune T cells from BALB/c mice immunized with anti-Id MAb 7.B12 (50 µg/ml) and stimulated with Ab2, gp43 or irrelevant MAb 1.H2. As negative control were used immune cells without antigenic stimulation (medium). As positive control Concanavalin A (Con A) was used. Proliferative response was determined 72 h later in a [3H]-thymidine incorporation assay. The results are representative of three independent experiments and are expressed as arithmetic means ± SD.

Fig. 6.

Fig. 6

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Proliferation of immune T cells from BALB/c mice immunized with irrelevant MAb 1.H2 (50 µg/ml) and stimulated with Ab2 or gp43. As negative control were used immune cells without antigenic stimulation (medium). As positive control Concanavalin A (Con A) was used. Proliferative response was determined 72 h later in a [3H]-thymidine incorporation assay. The results are representative of three independent experiments and are expressed as arithmetic means ± SD.

Fig. 7.

Fig. 7

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Production of IFN-γ by lymph nodes cells from mice immunized with Anti-Id MAb 7.B12 and stimulated in presence of gp43 (5 µg/ml), Ab2 (4 µg/ml) or irrelevant MAb 1.H2 (4 µg/ml). As negative control were used immune cells without antigenic stimulation (control). Results are representative of three independent experiments, and expressed as arithmetic means ± SD.

DISCUSSION

Lymphocyte antigen receptors (i.e. immunoglobulin on B cells or T cell receptor on T cells) express antigenic determinants that can be recognized and elicit a humoral or cellular immune response in xenogeneic, allogeneic, syngeneic or even in autologous animals [12–14,16]. Thus, foreign antibody molecules given to an individual or new antibodies arising in serum have the potential to induce immune responses, mainly directed to variable region determinants known collectively as idiotypes (Ids). Jerne's network theory predicts that anti-Id antibodies bearing the internal image of the corresponding antigen are able to induce immunity to original antigen in hosts not previously exposed to it [12,16,17]. It has been suggested that the immune response can be regulated at the level of recognition of idiotype determinants via the idiotype-anti-idiotype network [13,14,23]. While there is substantial evidence indicating that anti-idiotypic antibodies are normal components of the immune response, their significance in its regulation remains unclear [24,25]. Although it is generally assumed that anti-Id antibodies which arise in an intact network will reduce immune responses associated with that particular idiotype, anti-idiotypic networks may also have the capacity to increase immune response [26,27].

Anti-Ids that recognize a common Id and represent an internal image of the antigen have been generated in many systems [25,28]. Their antigenic mimicry makes then valuable as substitutes not only for certain hormones, e.g. gonadotropin [29], by binding to a specific cell receptor, but also for infectious agents as immunogens by eliciting a specific antibody response [30]. Development of anti-Ids as diagnostic reagents has not been achieved to the same extent. The approach of using anti-Ids as reagents has several advantages over current immunoassays. Since anti-Ids are immunoglobulins and not infectious, they overcome the inherent problems of working with dangerous animal and human pathogens, facilitate continuous production of the reagents by hybridoma technology and enhance the test reproducibility and standardization.

To elucidate whether MAb Ab2 could act as antigen surrogate in serological assays, serum from PCM patients were tested. All patients' sera were found positive for antigp43 antibody by capture enzyme immunoassay (EIA) using purified gp43. Using an ELISA test it was observed that antibodies from patients, but not normal serum, bind to Ab2, suggesting that MAb 7B12 could substitute gp43. The use of anti-Id as a diagnostic reagent was first reported in 1982 by Potocnjak et al. [31] for Plasmodium berghei, using an inhibition test in which the radiolabelled anti-Id competed with the antigen for binding to the solide-phase Id. A more recent study using anti-Ids as serodiagostic reagents for bovine cysticercosis employed anti-Id directly on the solid phase of ELISA plates and detected antibodies against Taenia parasite antigens at a level comparable to that employing parasite antigens on the solid phase [32]. Unfortunately, in our system cross-reactions with sera from patients with other mycoses were observed. In order to better understand those cross-reactions, a plate was coated with irrelevant MAb. The result showed that both, heterologous and PCM sera reacted with the irrelevant MAb. Moreover, no significant inhibition of ELISA assay was observed when heterologous sera were previously incubated with the deglycosylated form of gp43 (p38). These results suggest that the cross-reactions observed with 7B12 MAb were not related to the gp43 binding epitope. On the other hand, ELISA test using Ab2 bound to the solid phase made it possible to serologically monitor patients under antifungal therapy, since an equivalent curve with ELISA test using purified gp43 was obtained.

Cellular response is more crucial than humoral response in PCM. In this regard, the ability of anti-Id to evoke T-cell mediated anti-P. brasiliensis response may be more relevant to effective therapy than its ability to induce a humoral response. Thus, induction of protective cellular response should be included in the criteria to select anti-Id for PCM therapy.

Although the majority of anti-idiotypic antibodies have been used to stimulate antibody responses, they may also stimulate T cells. Unprimed T cells are stimulated by antigen-presenting cells that take up antigen and after processing, present it on their MHC molecules in combination with costimulatory signals. Anti-idiotypic antibodies may that recognize paratopes of anti-tumour antibodies act as internal images of the tumour antigen and so be used as surrogate antigens. Two anti-idiotypic antibodies, from two different experimental models,105D7 and 708, have been used to study T-cell reponses by anti-idiotypes [33,34]. 105D7 mimics CD55 and can stimulate both T-cell and antibody response that recognize tumour cells expressing CD55. 708 induce antibody responses that recognize CEA and can stimulate lymphocytes from colorectal cancer patients to respond to in vitro challenge with CEA. This anti-idiotype has been cloned and sequenced, disclosing peptide homology with CEA [35].

Since the antigen mimicry properties of Ab2β are species-independent, and mouse Ab2β is potentially suitable as a surrogate antigen in human PCM patients, in the present work it was also analysed whether Ab2β can induce T cell immunity. Our results showed that when mice were immunized whit Ab2β and exposed to gp43 in vitro, a specific T cell proliferation was obtained. It was also observed that these cells produced preferentially IFN-γ, a cytokine involved with protection in PCM. Recently, Taborda et al. [36] have shown that gp43-immunized mice led to an IFN-γ-mediated protection against intratracheal challenge with virulent P. brasiliensis and identified a 15-amino-acid peptide (p10) as T-cell-epitope. It is tempting to speculate that Ab2β bearing internal image of gp43 could contain the p10 T-cell epitope, which was responsible to induce lymphproliferation. The possibility that some short amino acid sequences could be part of conformational epitope and thus able to induce response against the whole epitope can not be ruled out. Future studies will determine the structural basis for gp43 mimicry by Ab2 by identifying the peptide(s) derived from the hypervariable region of the Ab2 that can induce specific humoral and/or cellular immunity to gp43.

Previous studies have shown that anti-idiotypes are superior to peptides or antigen in presenting viral epitopes to CD4 T cells [37]. Then, we suggest that Ab2β, that contain a T-cell epitope, could be used in a protection assay. Experiments are being carried out to elucidate this question.

These results open several possibilities for applications of Ab2β in serology and therapy. These findings could open new strategies for diagnosis and therapy of fungal infection without the necessity of handling the pathogenic microorganism.

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