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Trypanosoma cruzi: Involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5
EXPERIMENTAL
PARASITOLOGY
75,
137-145 (1992)
cruzi: Involvement of IgG lsotypes in the Parasitemia Control of Mice Immunized with Parasite Exoantigens of Isoelectric Point 4.5
Trypanosoma
FABIO CERBAN, ADRIANA GRUPPI, AND ELSA VOTTERO-CIMA Inmunologia,
Departamento
de Bioquimica Clinica, Facultad de Ciencias de Cordoba, Cordoba, Argentina
Quimicas,
Universidad
National
CERBAN, F., GRUPPI, A., AND VOTTERO-CIMA, E. Trypanosoma cruzi: Involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5. Experimental Parasitology 75, 137-145. In a previous work we demonstrated that Ttypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgGl antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgGl and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33°C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56°C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgGl and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgGl retains most of this capacity. Purified immune IgGl revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5. o IWZ Academic press. IW. INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Antibody isotypes; Glucidic epitopes; Parasitemia; Protection; Bordetella pertussis (BP); Exoantigens (Ea); Enzyme-linked immunosorbent assay (ELISA); Human serum albumin (HSA); Isoelectric focusing (IEF); Immediate-type hypersensitivity (ITH); Kilodaltons (kDa); Molecular weight (MW); Optical density (OD); Phosphate buffered-saline (PBS); Isoelectric point (PI); Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); Trypomastigotes (TP).
A major characteristic of parasitic infection is the presence of a variety of antigens in the circulation of human and other infected hosts (Targett 1982; NogueiraQueiroz et al. 1986; Asai et al. 1987). In regard to American trypanosomiasis, whose etiologic agent is the protozoan flagellate Trypanosoma cruzi, parasite-released antigens and antibodies against them have been detected in biological fluids in human and experimental infections (Araujo 1976; Gottlieb 1977; Araujo et al. 1981; Moretti et al. 1985; Freilij et al. 1987).
With reference to the function of released molecules, some of them may facilitate the penetration/migration of the parasite through the tissues (Castanys er al. 1990), others may divert inflammatory response away from the surface of the parasite or modulate the immunological response of the host. There is also some evidences of their ability to induce a protective immune response (Araujo et al. 1978; James 1989; Ouaissi et al. 1990). The immunization of mice with exoantigens of T. cruzi of ~14-5, prior to the infection, induced a protective immune response as judged by the levels of parasitemia and the diminished mortality 137 00144894192 $5.00 Copyright 0 1992 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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(Gruppi et al. 1991). The same effect was observed with the more purified exoantigens that focused at ~14.5 when these were injected with Bordetella pertussis as adjuvant (Cerban et al. 1991). Several immune mechanisms were proposed as responsible for the acquired resistance against T. cruzi. The participation of the antibody in the defence mechanisms has been reported by passive transfer of protection with immune serum from infected mice (Krettli and Brener 1976; Scott and Goss-Sampson 1984). Because of the reports mentioned above, the aim of this work was to study the participation of humoral murine immune response against Ea 4.5 on the parasitemia control in the infection with T. cruzi. We analysed the involvement of different isotypes of antibodies directed against Ea 4.5 and the participation of antibodies against the glucidic epitopes of Ea 4.5, the most relevant epitopes for humoral immune response (Cerban et al. 1991). MATERIALSANDMETHODS Parasites and mouse strains. The Tulahuen strain of T. cruzi was used. It was maintained by weekly intraperitoneal (ip) inoculations in 30- to 60-day-old BALB/c mice. Parasite antigens. Plasma, free from parasites, was obtained from infected mice as previously described (Moretti et al. 1985). It was used as a source of Ea’s. Briefly, plasma was obtained from T. cruzi-infected BALB/c mice 8 days after infection with lo6 trypomastigotes and is referred to as acute infection plasma. Ea 4.5 was purified from acute infection plasma by isoelectric focusing as described below. Isoelectric focusing. Isoelectric focusing (IEF) was performed in horizontal slab 1% agarose (Pharmacia Fine Chemicals, Uppsala, Sweden) gels prepared on glass slabs to the approximate dimensions of 0.1 x 11.5 X 12 cm. These gels contained 2.7% ampholines, pH 4-6.5, and 4.1% ampholines, pH 2.5-5. Samples of acute infection plasma (200 @slab) were applied to the gel and were focused at 600 V, 30 mA, 5 W for 3 hr. The unstained fraction of agarose-containing exoantigens focalized at pI 4.5 (see Fig. 1) was used as a source of Ea 4.5 for mice immunization. For immunoblotting, after electrophoresis, unstained gels were transferred to a nitrocelhtlose membrane (NC) (Schleicher & SchuIl) using the capillary
VOTTERO-CIMA PH 6.5
FIG. 1. Blotting of acute infection plasma separated by IEF, pH gradient 2.5-6.5, and then incubated with sera from chronic chagasic (line 1) and control (line 2) patients. The arrow indicates the position of the Ea 4.5. Fixation of labeled protein A at upper dark zone, observed on nitrocellulose sheets incubated with the control and chagasic sera samples, was due to mouse immunoglobulins. procedure previously described (Reinhart and Malamud 1982). The NC sheets were then incubated with sera from chagasic patients or control human sera at l/100 dilution in the probe binding buffer (Gruppi et al. 1990) for 16 hr at 4°C. Fixed IgG was revealed by incubation with ‘251-labeled Protein A followed by autoradiography with a 3 M X-ray film (Greenwood et al. 1963). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fraction of agarose containing Ea 4.5 (about 200 pg of proteins) was solubilized in sample buffer (0.06 Tris-CIH, pH 6.8; 4% SDS and 2% 2-mercaptoethanol), boiled for 3 min and applied to a 7-cm polyacrylamide gel 10% (w/v). The electrophoresis was performed as described by Laemmli (1970). Ea 4.5 bands were transferred from the separating gel to NC using the electrophoretic procedure previously described by Towbin et al. 1979. The antigens were detected by sequential addition of IgGl fraction from immune and control sera, then goat antimouse IgGl (Sigma) and peroxidase-labeled rabbit anti-goat (Sigma), and finally, H,O, plus Cchloro-lnaphthol as substrate. The reaction was stopped by washing with distilled water. Animal immunizations. BALBlc mice, 2 months old (n = 6), were immunized intradermally (id) with three weekly doses of Ea 4.5 antigen (about 100 &dose). Inactivated Bordetella pertussis (BP), strain 10536 (Instituto National de Microbiologia “Carlos Malbran”, Buenos Aires) was used as adjuvant in a concentration
ANTI-T.
Cruzi EXOANTIGENS
AND CONTROL
of 1.25 x lo9 U per mouse per dose (Rottemberg et al. 1988). Control animals (n = 6) were injected with agarose taken from p1 4.5 of a gel loaded with normal mouse plasma plus Bp. Two weeks after the last dose, the immediate-type hypersensitivy (ITH) was assayed and sera from all animals were obtained for antibody assays and transfer experiments. Immediate-type hypersensitivity. ITH was performed by a challenge with 50 ul of PBS containing 50 ug of Ea 4.5 into a hind footpad. As control, 50 ~1 of PBS-containing material obtained from the same p1 zone of normal mouse plasma separated by IEF was injected into the contralateral footpad. The thickness of both the antigen and control injected footpads was measured with a dial micrometer 20 mitt after the inoculation. The results are expressed as the difference between the thickness of antigen (Ea 4.5) and control inoculated footpads (Pistoresi-Palencia et al. 1984). Isolation of murine IgG isotypes by afJinity chromatography with protein A-Sepharose. Protein A-bound Sepharose CL-4B (Pharmacia Fine Chemicals) was resuspended in 0.15 M PBS, pH 7.2, and mounted in a 1 X 5-cm column. This column was equilibrated with 1.5 M glycine-3 M ClNa buffer, pH 8.9. An aliquot (1 ml) of immune or control sera was loaded onto the column and the nonadsorbed proteins washed off with 1.5 M glycine3 M ClNa buffer, pH 8.9. The adsorbed proteins were then stepwise eluted using buffer of different pH as described by Ey et al. (1978), following the Pharmacia instructions (Separation News, Vol. 13/5). IgGl was eluted with 0.1 M citrate buffer, pH 6.0, whereas IgG2 was eluted with 0.1 M citrate buffer, pH 5.0. Samples (3 ml) were collected, and fractionation was monitored by measurements of optical density at 280 nm. The eluates of each protein peak were pooled separately, dyalized against PBS, and concentrated to the original serum volume. The overlap between the different isotypes detected by ELISA was eliminated by absorption with goat anti-mouse IgG2 and antimouse IgG3 for eluate containing IgGl. In the same way, the eluate containing IgG2 was absorbed with goat anti-mouse IgGl and anti-mouse IgG3. Treatment of sera at 56’C. The immune and control sera were maintained at 56°C for 2 hr before the incubation with the bloodstream trypomastigotes (Tp). Antibody Assays. For this test, the components of Ea 4.5 fraction were eluted from IEF gel with absorption buffer for ELISA (0.05 M carbonate-bicarbonate, pH 9.6) and concentrated to 100 &ml. Polystyrene plates were coated with Ea 4.5 or with formaldehydetreated epimastigotes (EPI) in a concentration of 10’ parasites in PBS/well overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBS-Tweet& the plates were blocked with PBS-Tween containing 5% human serum albumin (PBS-Tween-HSA) for 60 mitt at 37°C. Plates were incubated for 60 min at 37°C with set-a from the immunized and control animals diluted in PBS-Tween-HSA. Plates were washed
OF PARASITEMIA
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against PBS-Tween. For the analysis of IgG isotypes, goat anti-mouse isotypes (IgGl, IgG2, and IgG3; Sigma Chemical Co) were used followed by the perox&se-labeled rabbit anti-goat IgG (l/500 in PBSTween-HSA). Plates were read at 492 nm after incubation with H,O, and o-phenylendiamine. The mean of absorbancy of duplicates was used for statistical analysis. Values were considered positive when the OD obtained by diluted inmune sera were higher than the mean +2 SD of OD obtained by diluted control sera (n = 6). Absorption of immune sera with the glucidic moeity of Ea 4.5. The aqueous phase (100 ul) obtained from Ea 4.5 (100 pi/ml) by phenol-water extraction (Westphal and Jann 1%5) diluted in carbonatebicarbonate buffer was used to coat polystyrene plates overnight at 4°C. Before washing with PBS-Tween, the plates were blocked with PBS-Tween-HSA for 30 min at 37°C. For the absorption, undiluted immune sera (100 ~1) were added and maintained for 30 min at 37°C. The absorption was repeated five times. Then the sera were collected and the diminution of reactivity to Ea 4.5 carbohydrate was controlled by ELISA. The absorbancy at 492 nm of control, immune, and absorbed immune pooled sera, at l/50 dilution, were 0.112, 0.491, and 0.197, respectively. Mouse infection with Tp preincubated with Eu 4.5 antibodies (transfer experiments). Undiluted immune or control sera were mixed with an equal volume (50 ~1) of suspension of Tp, isolated from mouse blood from the orbital sinuses. The Tp were then diluted with PBS to a concentration of lo?50 pJ. The mixture was incubated at 33°C for 1 hr. The morphology and viability of Tp were examined under light microscopy. Normal syngeneic mice (n = 10) were inoculated with 100 pl of the Tptreated suspension by intraperitoneal route. The course of the parasitemia was determined periodically. In the same way, the parasitemia was analysed in groups of animals (n = 10) infected with Tp without treatment or incubated with the following preparations: (1) immune and control sera heated at 56°C for 2 hr, (2) immune sera absorbed with the carbohydrate moeity of Ea 4.5, (3) IgGl purified from immune and control sera, and (4) IgG2 purified from immune and control sera. Parasitemia. The parasitemia was periodically determined starting on Day 10 postinfection. Ten microliters of blood samples were taken from the tails of infected mice, mixed with 90 pl 0.85% ammonium chloride to lyse red cells, and then the parasites were counted in a Neubauer chamber. Statistical analysis. Statistical analysis was performed using Student’s t test. RESULTS
In order to study the participation of antibody isotypes on the parasitemia control
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CERRAN , GRUPPI , AND VOTTERO-CIMA
in the infection with T. cruzi, we determined the IgG isotypes elicited in mice immunized id with Ea 4.5 (see Fig. 1) mixed with Bp as adjuvant. Sera of these mice obtained 2 weeks after the last immunization, assayed by ELISA using Ea 4.5~coated plates, showed that IgGl was the main isotype produced, but binding of IgGl and IgG2 antibodies was observed when formaldehyde-fixed EPI were used as antigen for the test (Fig. 2). The ability of Ea 4.5 to induce ITH response against the homologous antigen was detected on Day 15 after the last immunization (P < O&01), (Fig. 3), indicating the presence of reaginic antibodies (IgE and/or IgGl) specific for Ea 4.5. Mouse infection with Tp preincubated with Ea 4.5 antibodies. To study the in-
volvement of the immune humoral response induced by Ea 4.5 in the control of parasitemia, we analysed the effect of immune sera on the Tp of T. cruzi. The Tp preincubated with anti-Ea 4.5 sera for 1 hr at 33°C showed no significant changes in their morphology and mobility. These Tp ip injected into normal singeneic mice produced parasitemia levels lower than that of those preincubated with sera of the controls (P < 0.01, Day 10 postinfec___----
6 Swalling --~--__-----of footpad x 1110 mm _______
r
0
---
Day 15 After the last immunization
FIG. 3. Specific ITH response in mice immunized with Ea 4.5. Each bar represents the difference between the thickness of antigen (Ea 4.5) and controlinoculated footpads. n , Control; K& immune.
tion; P < 0.001, Day 12 postinfection; p < 0.005, Day 14 postinfection) (Fig. 4). In order to analyse the involvement of isotype immunoglobulins sensitive at 56°C IgE (Damonneville et al. 1986) and IgG2a (Wechsler and Kongshavn 1986) sera from immune mice and controls were kept for 2 hr at 56°C before incubation with Tp. This treatment did not modify the anti-Tp activity of the anti-Ea 4.5 sera (P < 0.005, Day x lO+/ml mr Parasites _____ -___-----------------
1
Days post infection FIG. 2. IgG isotype profdes of antibodies induced by immunization with Ea 4.5. Antigens used for the ELISA tests, Ea 4.5 (m) and EPI (a). Each bar represents the mean of the titres + SD.
FIG. 4. Parasitemia of mice infected with Id Tp of T. cruzi preincubated with immune and control sera on Days 10, 12, and 14 postinfection. n , Control; El, immune.
ANTI-T.
Cf-UZi
EXOANTIGENS
10 postinfection; P < 0.025, Day 12 postinfection) (Fig. 5). In addition, to study the involvement of Ea 4.5 glucidic epitopes, sera from immune mice were absorbed with the glucidic fraction obtained from Ea 4.5 before incubation with the Tp. Such absorption diminished the anti-Tp capacity of the anti-Ea 4.5 sera (P < 0.05, days 12 and 14 postinfection), (Fig. 6). Effect on the Tp of ZgGl and ZgG2 purified from mouse anti-Ea 4.5 sera. The Tp
preincubated with IgGl antibodies and used to infect normal mice yielded levels of parasitemia significantly lower than those of the Tp preincubated with IgGl from the controls (P < 0.005, Day 12 postinfection; P < 0.001, Day 14 postinfection). In contrast, such effect was not observed in the groups preincubated with IgG2 from immune or control sera (Figs. 7A, 7B, 7C). Molecular weight of T. cruzi Ea 4.5 recognized by mouse ZgGl fraction. To define the molecular weight (MW) of T. cruzi Ea 4.5 recognized by IgGl fraction from immunized mice, SDS-PAGE using Ea 4.5 previously separated by IEF was performed, followed by Western blotting. As can be
Parasites
r
25 --
x lO+/ml
I
1
Days post infection
FIG. 5. Effect of treatment of immune and control sera at 56°C for 2 hr. Parasitemia of mice infected with lo3 Tp preincubated with immune- and control-treated sera on Days 10 and 12 postinfection. n , Control; H, immune.
AND
CONTROL
OF
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PARASITEMIA
x IO-5/ml $0 Parasites --------------
------
------,
I-
!
Dayspost infection FIG. 6. Effect of absorption of immune sera with the glucidic moeity of Ea 4.5. Parasitemia of mice infected with lo3 Tp preincubated with control (W), immune (H), and absorbed immune (Cl) sera on Days 12 and 14 postinfection.
seen in Fig. 8, the IgGl fraction revealed two antigenic bands of MW between 50 and 55 kDa. DISCUSSION
We have previously demonstrated that circulating antigens released by T. cruzi share epitopes with parasite cell surface (Gruppi et al. 1989; Cerban et al. 1991). On the other hand, some experimental evidence shows that membrane antigens can induce a protective immune response (Scott and Snary 1979; Champsi and McMahon-Pratt 1988; Norris et al. 1989). Mouse immunization with Ea 4.5 and Bp as adjuvant produced resistance to T. cruzi infection as revealed by a significantly lower parasitemia and a longer survival of mice immunized compared to control animals (CerbBn et al. 1991). Since the effectiveness of humoral immunity against parasites may depend on the presence of antigen epitopes which bind antibody isotypes capable of mediating protective reactions (Vignali et al. 1989), in this paper we studied the involvement of anti-Ea 4.5 antibody isotypes. Mouse immunization with Ea 4.5 and Bp as adjuvant induced specific IgG with predominance of IgGl. The IgG2 isotype could be
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, GRUPPI
, AND
VOTTERO-CIMA
looParasitssx 10~%ml
--T--l
b
,,J%fss
x 10-5/ml
--__ I
60.
Days post infection
IL Days pcstinfection
‘-
FIG. 7. Parasitemia of mice infected with 10’ Tp preincubated (A) without sera or with immune and control sera, (B) with immune and control IgGl, (C) with immune and control IgG2, on Days 12 and 14 postinfection. n , Control sera; H, immune sera; q , without sera.
detected only by using EPI as antigen in the ELISA test. In addition, ITH was detected, indicating the presence of reaginic antibodies (IgE and/or IgGl) specific for Ea 4.5. Some authors working with sera from mice chronically infected with Tp of T. cruzi (Takehara ef al. 1981; Brodskyn et al. 1988) or with dead EPI plus complete Freund’s adjuvant (Scott and Goss-Sampson 1984) demonstrated a predominance of specific IgG2 antibodies. In this work the specific IgGl isotype was mainly elicited by K
55 I-) 50-t
FIG. 8. SDS-PAGE of Ea 4.5 followed by Western blotting using immune igG1 (line 1) and control IgGl fraction (line 2). The arrows indicate the position of the bands. The MW of markers from top to bottom are: 94, 67,43, 30, 20.1, and 14.4 kilodaltons &Da).
using a soluble antigen released by T. cruzi (Ea 4.5), Bp as adjuvant, and the immunization schedule already indicated. It is known that humoral immune response to certain antigens is often biased towards the production of particular immunoglobulin isotype (Larsson et al. 1975; Perlmutter et aZ. 1978; Stevens et al. 1983; Callard and Turner 1990). Moreover, the addition of the adjuvant may also contribute to this (Monroy et al. 1989; Karagouni and HadjipetrouKouronakis 1990). Ea 4.5-Bp might result in activation of effector helper cells which would preferentially act on Ea 4.5~specific B cells that have already switched with surface IgGl. This isotype seems to be involved in the parasitemia control of mice challenged with Tp after immunization. The results obtained with treatment of Tp with anti-Ea 4.5 sera indicate that the humoral immunity against Ea 4.5 may play a role in the control of parasitemia in the mice infected postimmunization. This activity was resistant at 56°C suggesting that, in the immunization schedule assayed, the IgGl isotype is the main antibody involved since the other isotypes that may be involved (IgE and IgG2a) are sensitive at this temperature (Damonneville et al. 1986; Weschsler and Kongshavn 1986). In addition, experiments carried out with IgGl and IgG2 purified from immune sera showed
ANTI-T.
Ct74Zi
EXOANTIGENS
AND
that only IgGl isotype was able to decrease the levels of parasitemia in this experimental system. These findings are in agreement with Brodskyn et al. (1989), who obtained some evidences of the ability of IgGl to perform the clearance of parasite bloodstream forms. Furthermore, the ability of parasitemia control of IgGl can now be added to that of IgG2 (Takehara et al. 1981; Scott and GossSampson 1984; Brodskyn et al. 1988) induced during the infection because the parasite can have epitopes that are absent in the Ea 4.5 and which may be able to induce the IgG2 isotype. Even though IgG2 was considered the protector antibody according to the tindings of Takehara et al. (1981) and Brodskyn et al. (1988), it was recently demonstrated that the low-affinity receptor (FcyRII), to which IgGl binds better than IgG2a, becomes a high-affinity receptor by the effect of proteases (van de Winkel et al. 1989), particularly abundant in inflamation sites. The proteases can be released from macrophages after interaction with immune complexes (Cardella et al. 1974) and activated T cells could be another source of proteolytic enzymes (Fruth et al. 1987). Thus, IgGl could efficiently participate in the antibodydependent cellular cytotoxicity or phagocytosis through a receptor different from that of IgG2a and may contribute to the defense mechanisms of the host infected with T. cruzi. Studies about the specificity of the IgGl anti-Ea 4.5 revealed the binding of two components of MW between 50 and 55 kDa. Moreover, it was recently reported that the most relevant epitopes of Ea 4.5 are carbohydrates (Cerban et al. 1991), so we assayed the involvement of antibodies against carbohydrates from Ea 4.5 in the clearance of T. cruzi. It was seen that this capacity was diminished by absorption of immune sera with the glucidic fraction of Ea 4.5. This finding confirms the relevant
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role played by the glucidic epitopes of Ea 4.5, showing that antibodies against these epitopes are important in controlling parasitemia. Further studies will aim to explain how the Ea 4.5 carbohydrate-specific IgGl antibodies interact to eliminate the parasites from T. cruzi-infected mice. ACKNOWLEDGMENTS
The authors are grateful to Dr. Horatio Serra for providing protein A-Sepharose CL-4B and to The Instituto National de Microbiologia “Carlos Malbran” (Buenos Aires) for providing Bordetella pertussis. This work was supported by The Consejo de Investigaciones CientiIicas y Tecnologicas de la Provincia de Cordoba, (CONICOR), Argentina. F. C. thanks CONICOR and A. G. thanks The Consejo National de Investigaciones Cientiticas y Tecnicas (CONICET), Argentina for the fellowships granted. REFERENCES
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KARAGOUNI, E. E., AND HADJIPETROU-KOURONAKIS, L. 1990. Regulation of isotype immunoglobulin production by adjuvants in vivo. Scandinavian Journal of Immunology 31, 745-754. KRETTLI, A. U., AND BRENER, Z. 1976. Protective effects of specific antibodies in Trypanosoma cruzi infection. Journal of Immunology 166, 755-760. LAEMMLI, U. K. 1970. Clearance of structural proteins during the assembly of the head of bactetiophage T4. Nature 227, 680485. LARSSON, A., PISSARI-SALSANO, S., OHLANDER, C., NATVIG, J. B., AND PERLMANN, P. 1975. Destruction of dextran-coated target cells by normal lymphocytes and monocytes. Induction by human antidextran serum with IgG antibodies restricted to the IgG2 subclass. Scandinavian Journal of Immunology 4, 241-252. MONROY, F. G., ADAMS, J. H., DOBSON, C., AND EAST, I. J. 1989. Nemastospiroides dubuis: Iniluence of adjuvant on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult forms. Experimental Parasitology 68,6773. MORETTI, E. R. A., BASSO, B., AND VOTTERO-CIMA, E. 1985. Exoantigens of Trypanosoma cruzi. I. Conditions for their detection and immunogenic properties in experimental infection. Journal of Protozoology 32, 150-153. NOGUEIRA-QUEIROZ, J. A., LUTSCH, C., CAPRON, M., DESSAINT, J. P., AND CAPRON, A. 1986. Detection and quantification of circulating antigen in schistosomiasis by a monoclonal antibody with immunodiagnostic. Clinical and Experimental Immunology 65, 223-23 1. NORRIS, K. A., HARTH, G., AND So, M. 1989. Puritication of a Trypanosoma cruzi membrane glycoprotein which elicits lytic antibodies. Infection and Immunity
51, 2372-2377.
OUAISSI, M. A., TAIBI, A., CORNETTE, J., VELGE, P., MARTY, B., LOYENS, M., ESTEVA, M., RIZVI, F. S., AND CAPRON, A. 1990. Characterization of major surface and excretory-secretory immunogens of Trypanosoma cruzi trypomastigotes and identifi-
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AND
cation of potential protective antigen. Parasitology 100, 115-124. PERLMUTTER, R. M., HAMBURG, D., BRILES, D. E., NICOLOTTI,
R. A.,
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DAVIE,
J. M.
1978.
Sub-
class restriction of murine anti-carbohydrate antibodies. Journal of Immunology 121, 566-572. PISTORESI-PALENCIA, M. C., RIERA, C. M., GALMARINI,
M.,
VOTTERO-CIMA,
E.,
AND
SERRA,
H. M. 1984. Study of autoimmune response to rat male accesory glands in rats born to immune mothers. Immunology 53, 141-146. REINHART, M. P., AND MALAMUD, D. 1982. Protein transfer from isoelectric focusing gels: the native blot. Analytical Biochemistry 123, 229-235. ROTTEMBERG, M. E., CARDONI, R. L., DE TITTO, E. H., MORENO, M., AND SEGURA, E. 1988. Trypanosoma cruzi: Immune response in mice immunized with parasite antigens. Experimental Parasitology 65, 101-108. SCOTT, M. T., AND GOSS-SAMPSON, M. 1984. Restricted IgG isotype profiles in Trypanosoma cruzi infected mice and Chagas’ disease patients. Clinical and Experimental
Immunology
58, 372-379.
Scorr, M. T., AND SNARY, D. 1979. Protective immunization of mice using cell surface glycoprotein from Trypanosoma STEVENS,
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R., DICHEK,
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3, 6549.
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H. A., PERINI, A., SILVA, M. H., AND 1981. Trypanosoma cruzi: Role of different antibody classes in protection against infection in the mouse. Experimental Parasitology 52, 137-146. TARGETT, G. A. T. 1982. Immunology of Trypanosomes. In “Immunology of human infection” (A. Nahmias and R. O’Reilly, Eds.), pp. 45w86. Plenum, New York. TOWIN, H., STAHELIN, T., AND GORDON, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proceedings of
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W. J. 1989. Proteolisis induces increased binding affinity of monocyte type II FcR for human IgG. Journal of Immunology VIGNALI, D. A. A.,
143, 571-578. BICKLE, Q. D., AND TAYLOR, M. G. 1989. Immunity to Schistosoma mansoni in vivo: Contradiction or clarification? Immunology Today 10, 410-416. WESCHLER, D. S., AND KONGSHAVN, P. A. L. 1986. Heat-labile IgG2a antibodies affect cure of Trypanosoma musculi infection in CSBLl6 mice. Journal of Immunology 137, 2968-2972. WESTPHAL, O., AND JANN, K. 1965. Bacterial li-
popolysaccharides extraction with phenol-water and further applications of the procedure. Methods in Carbohydrate Chemistry 5, 83-91. Received 13 December 1991; accepted with revision 14 April 1992.
PARASITOLOGY
75,
137-145 (1992)
cruzi: Involvement of IgG lsotypes in the Parasitemia Control of Mice Immunized with Parasite Exoantigens of Isoelectric Point 4.5
Trypanosoma
FABIO CERBAN, ADRIANA GRUPPI, AND ELSA VOTTERO-CIMA Inmunologia,
Departamento
de Bioquimica Clinica, Facultad de Ciencias de Cordoba, Cordoba, Argentina
Quimicas,
Universidad
National
CERBAN, F., GRUPPI, A., AND VOTTERO-CIMA, E. Trypanosoma cruzi: Involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5. Experimental Parasitology 75, 137-145. In a previous work we demonstrated that Ttypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgGl antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgGl and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33°C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56°C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgGl and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgGl retains most of this capacity. Purified immune IgGl revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5. o IWZ Academic press. IW. INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Antibody isotypes; Glucidic epitopes; Parasitemia; Protection; Bordetella pertussis (BP); Exoantigens (Ea); Enzyme-linked immunosorbent assay (ELISA); Human serum albumin (HSA); Isoelectric focusing (IEF); Immediate-type hypersensitivity (ITH); Kilodaltons (kDa); Molecular weight (MW); Optical density (OD); Phosphate buffered-saline (PBS); Isoelectric point (PI); Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); Trypomastigotes (TP).
A major characteristic of parasitic infection is the presence of a variety of antigens in the circulation of human and other infected hosts (Targett 1982; NogueiraQueiroz et al. 1986; Asai et al. 1987). In regard to American trypanosomiasis, whose etiologic agent is the protozoan flagellate Trypanosoma cruzi, parasite-released antigens and antibodies against them have been detected in biological fluids in human and experimental infections (Araujo 1976; Gottlieb 1977; Araujo et al. 1981; Moretti et al. 1985; Freilij et al. 1987).
With reference to the function of released molecules, some of them may facilitate the penetration/migration of the parasite through the tissues (Castanys er al. 1990), others may divert inflammatory response away from the surface of the parasite or modulate the immunological response of the host. There is also some evidences of their ability to induce a protective immune response (Araujo et al. 1978; James 1989; Ouaissi et al. 1990). The immunization of mice with exoantigens of T. cruzi of ~14-5, prior to the infection, induced a protective immune response as judged by the levels of parasitemia and the diminished mortality 137 00144894192 $5.00 Copyright 0 1992 by Academic Press, Inc. AU rights of reproduction in any form reserved.
138
CERBAN,GRUPPI,AND
(Gruppi et al. 1991). The same effect was observed with the more purified exoantigens that focused at ~14.5 when these were injected with Bordetella pertussis as adjuvant (Cerban et al. 1991). Several immune mechanisms were proposed as responsible for the acquired resistance against T. cruzi. The participation of the antibody in the defence mechanisms has been reported by passive transfer of protection with immune serum from infected mice (Krettli and Brener 1976; Scott and Goss-Sampson 1984). Because of the reports mentioned above, the aim of this work was to study the participation of humoral murine immune response against Ea 4.5 on the parasitemia control in the infection with T. cruzi. We analysed the involvement of different isotypes of antibodies directed against Ea 4.5 and the participation of antibodies against the glucidic epitopes of Ea 4.5, the most relevant epitopes for humoral immune response (Cerban et al. 1991). MATERIALSANDMETHODS Parasites and mouse strains. The Tulahuen strain of T. cruzi was used. It was maintained by weekly intraperitoneal (ip) inoculations in 30- to 60-day-old BALB/c mice. Parasite antigens. Plasma, free from parasites, was obtained from infected mice as previously described (Moretti et al. 1985). It was used as a source of Ea’s. Briefly, plasma was obtained from T. cruzi-infected BALB/c mice 8 days after infection with lo6 trypomastigotes and is referred to as acute infection plasma. Ea 4.5 was purified from acute infection plasma by isoelectric focusing as described below. Isoelectric focusing. Isoelectric focusing (IEF) was performed in horizontal slab 1% agarose (Pharmacia Fine Chemicals, Uppsala, Sweden) gels prepared on glass slabs to the approximate dimensions of 0.1 x 11.5 X 12 cm. These gels contained 2.7% ampholines, pH 4-6.5, and 4.1% ampholines, pH 2.5-5. Samples of acute infection plasma (200 @slab) were applied to the gel and were focused at 600 V, 30 mA, 5 W for 3 hr. The unstained fraction of agarose-containing exoantigens focalized at pI 4.5 (see Fig. 1) was used as a source of Ea 4.5 for mice immunization. For immunoblotting, after electrophoresis, unstained gels were transferred to a nitrocelhtlose membrane (NC) (Schleicher & SchuIl) using the capillary
VOTTERO-CIMA PH 6.5
FIG. 1. Blotting of acute infection plasma separated by IEF, pH gradient 2.5-6.5, and then incubated with sera from chronic chagasic (line 1) and control (line 2) patients. The arrow indicates the position of the Ea 4.5. Fixation of labeled protein A at upper dark zone, observed on nitrocellulose sheets incubated with the control and chagasic sera samples, was due to mouse immunoglobulins. procedure previously described (Reinhart and Malamud 1982). The NC sheets were then incubated with sera from chagasic patients or control human sera at l/100 dilution in the probe binding buffer (Gruppi et al. 1990) for 16 hr at 4°C. Fixed IgG was revealed by incubation with ‘251-labeled Protein A followed by autoradiography with a 3 M X-ray film (Greenwood et al. 1963). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fraction of agarose containing Ea 4.5 (about 200 pg of proteins) was solubilized in sample buffer (0.06 Tris-CIH, pH 6.8; 4% SDS and 2% 2-mercaptoethanol), boiled for 3 min and applied to a 7-cm polyacrylamide gel 10% (w/v). The electrophoresis was performed as described by Laemmli (1970). Ea 4.5 bands were transferred from the separating gel to NC using the electrophoretic procedure previously described by Towbin et al. 1979. The antigens were detected by sequential addition of IgGl fraction from immune and control sera, then goat antimouse IgGl (Sigma) and peroxidase-labeled rabbit anti-goat (Sigma), and finally, H,O, plus Cchloro-lnaphthol as substrate. The reaction was stopped by washing with distilled water. Animal immunizations. BALBlc mice, 2 months old (n = 6), were immunized intradermally (id) with three weekly doses of Ea 4.5 antigen (about 100 &dose). Inactivated Bordetella pertussis (BP), strain 10536 (Instituto National de Microbiologia “Carlos Malbran”, Buenos Aires) was used as adjuvant in a concentration
ANTI-T.
Cruzi EXOANTIGENS
AND CONTROL
of 1.25 x lo9 U per mouse per dose (Rottemberg et al. 1988). Control animals (n = 6) were injected with agarose taken from p1 4.5 of a gel loaded with normal mouse plasma plus Bp. Two weeks after the last dose, the immediate-type hypersensitivy (ITH) was assayed and sera from all animals were obtained for antibody assays and transfer experiments. Immediate-type hypersensitivity. ITH was performed by a challenge with 50 ul of PBS containing 50 ug of Ea 4.5 into a hind footpad. As control, 50 ~1 of PBS-containing material obtained from the same p1 zone of normal mouse plasma separated by IEF was injected into the contralateral footpad. The thickness of both the antigen and control injected footpads was measured with a dial micrometer 20 mitt after the inoculation. The results are expressed as the difference between the thickness of antigen (Ea 4.5) and control inoculated footpads (Pistoresi-Palencia et al. 1984). Isolation of murine IgG isotypes by afJinity chromatography with protein A-Sepharose. Protein A-bound Sepharose CL-4B (Pharmacia Fine Chemicals) was resuspended in 0.15 M PBS, pH 7.2, and mounted in a 1 X 5-cm column. This column was equilibrated with 1.5 M glycine-3 M ClNa buffer, pH 8.9. An aliquot (1 ml) of immune or control sera was loaded onto the column and the nonadsorbed proteins washed off with 1.5 M glycine3 M ClNa buffer, pH 8.9. The adsorbed proteins were then stepwise eluted using buffer of different pH as described by Ey et al. (1978), following the Pharmacia instructions (Separation News, Vol. 13/5). IgGl was eluted with 0.1 M citrate buffer, pH 6.0, whereas IgG2 was eluted with 0.1 M citrate buffer, pH 5.0. Samples (3 ml) were collected, and fractionation was monitored by measurements of optical density at 280 nm. The eluates of each protein peak were pooled separately, dyalized against PBS, and concentrated to the original serum volume. The overlap between the different isotypes detected by ELISA was eliminated by absorption with goat anti-mouse IgG2 and antimouse IgG3 for eluate containing IgGl. In the same way, the eluate containing IgG2 was absorbed with goat anti-mouse IgGl and anti-mouse IgG3. Treatment of sera at 56’C. The immune and control sera were maintained at 56°C for 2 hr before the incubation with the bloodstream trypomastigotes (Tp). Antibody Assays. For this test, the components of Ea 4.5 fraction were eluted from IEF gel with absorption buffer for ELISA (0.05 M carbonate-bicarbonate, pH 9.6) and concentrated to 100 &ml. Polystyrene plates were coated with Ea 4.5 or with formaldehydetreated epimastigotes (EPI) in a concentration of 10’ parasites in PBS/well overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBS-Tweet& the plates were blocked with PBS-Tween containing 5% human serum albumin (PBS-Tween-HSA) for 60 mitt at 37°C. Plates were incubated for 60 min at 37°C with set-a from the immunized and control animals diluted in PBS-Tween-HSA. Plates were washed
OF PARASITEMIA
139
against PBS-Tween. For the analysis of IgG isotypes, goat anti-mouse isotypes (IgGl, IgG2, and IgG3; Sigma Chemical Co) were used followed by the perox&se-labeled rabbit anti-goat IgG (l/500 in PBSTween-HSA). Plates were read at 492 nm after incubation with H,O, and o-phenylendiamine. The mean of absorbancy of duplicates was used for statistical analysis. Values were considered positive when the OD obtained by diluted inmune sera were higher than the mean +2 SD of OD obtained by diluted control sera (n = 6). Absorption of immune sera with the glucidic moeity of Ea 4.5. The aqueous phase (100 ul) obtained from Ea 4.5 (100 pi/ml) by phenol-water extraction (Westphal and Jann 1%5) diluted in carbonatebicarbonate buffer was used to coat polystyrene plates overnight at 4°C. Before washing with PBS-Tween, the plates were blocked with PBS-Tween-HSA for 30 min at 37°C. For the absorption, undiluted immune sera (100 ~1) were added and maintained for 30 min at 37°C. The absorption was repeated five times. Then the sera were collected and the diminution of reactivity to Ea 4.5 carbohydrate was controlled by ELISA. The absorbancy at 492 nm of control, immune, and absorbed immune pooled sera, at l/50 dilution, were 0.112, 0.491, and 0.197, respectively. Mouse infection with Tp preincubated with Eu 4.5 antibodies (transfer experiments). Undiluted immune or control sera were mixed with an equal volume (50 ~1) of suspension of Tp, isolated from mouse blood from the orbital sinuses. The Tp were then diluted with PBS to a concentration of lo?50 pJ. The mixture was incubated at 33°C for 1 hr. The morphology and viability of Tp were examined under light microscopy. Normal syngeneic mice (n = 10) were inoculated with 100 pl of the Tptreated suspension by intraperitoneal route. The course of the parasitemia was determined periodically. In the same way, the parasitemia was analysed in groups of animals (n = 10) infected with Tp without treatment or incubated with the following preparations: (1) immune and control sera heated at 56°C for 2 hr, (2) immune sera absorbed with the carbohydrate moeity of Ea 4.5, (3) IgGl purified from immune and control sera, and (4) IgG2 purified from immune and control sera. Parasitemia. The parasitemia was periodically determined starting on Day 10 postinfection. Ten microliters of blood samples were taken from the tails of infected mice, mixed with 90 pl 0.85% ammonium chloride to lyse red cells, and then the parasites were counted in a Neubauer chamber. Statistical analysis. Statistical analysis was performed using Student’s t test. RESULTS
In order to study the participation of antibody isotypes on the parasitemia control
140
CERRAN , GRUPPI , AND VOTTERO-CIMA
in the infection with T. cruzi, we determined the IgG isotypes elicited in mice immunized id with Ea 4.5 (see Fig. 1) mixed with Bp as adjuvant. Sera of these mice obtained 2 weeks after the last immunization, assayed by ELISA using Ea 4.5~coated plates, showed that IgGl was the main isotype produced, but binding of IgGl and IgG2 antibodies was observed when formaldehyde-fixed EPI were used as antigen for the test (Fig. 2). The ability of Ea 4.5 to induce ITH response against the homologous antigen was detected on Day 15 after the last immunization (P < O&01), (Fig. 3), indicating the presence of reaginic antibodies (IgE and/or IgGl) specific for Ea 4.5. Mouse infection with Tp preincubated with Ea 4.5 antibodies. To study the in-
volvement of the immune humoral response induced by Ea 4.5 in the control of parasitemia, we analysed the effect of immune sera on the Tp of T. cruzi. The Tp preincubated with anti-Ea 4.5 sera for 1 hr at 33°C showed no significant changes in their morphology and mobility. These Tp ip injected into normal singeneic mice produced parasitemia levels lower than that of those preincubated with sera of the controls (P < 0.01, Day 10 postinfec___----
6 Swalling --~--__-----of footpad x 1110 mm _______
r
0
---
Day 15 After the last immunization
FIG. 3. Specific ITH response in mice immunized with Ea 4.5. Each bar represents the difference between the thickness of antigen (Ea 4.5) and controlinoculated footpads. n , Control; K& immune.
tion; P < 0.001, Day 12 postinfection; p < 0.005, Day 14 postinfection) (Fig. 4). In order to analyse the involvement of isotype immunoglobulins sensitive at 56°C IgE (Damonneville et al. 1986) and IgG2a (Wechsler and Kongshavn 1986) sera from immune mice and controls were kept for 2 hr at 56°C before incubation with Tp. This treatment did not modify the anti-Tp activity of the anti-Ea 4.5 sera (P < 0.005, Day x lO+/ml mr Parasites _____ -___-----------------
1
Days post infection FIG. 2. IgG isotype profdes of antibodies induced by immunization with Ea 4.5. Antigens used for the ELISA tests, Ea 4.5 (m) and EPI (a). Each bar represents the mean of the titres + SD.
FIG. 4. Parasitemia of mice infected with Id Tp of T. cruzi preincubated with immune and control sera on Days 10, 12, and 14 postinfection. n , Control; El, immune.
ANTI-T.
Cf-UZi
EXOANTIGENS
10 postinfection; P < 0.025, Day 12 postinfection) (Fig. 5). In addition, to study the involvement of Ea 4.5 glucidic epitopes, sera from immune mice were absorbed with the glucidic fraction obtained from Ea 4.5 before incubation with the Tp. Such absorption diminished the anti-Tp capacity of the anti-Ea 4.5 sera (P < 0.05, days 12 and 14 postinfection), (Fig. 6). Effect on the Tp of ZgGl and ZgG2 purified from mouse anti-Ea 4.5 sera. The Tp
preincubated with IgGl antibodies and used to infect normal mice yielded levels of parasitemia significantly lower than those of the Tp preincubated with IgGl from the controls (P < 0.005, Day 12 postinfection; P < 0.001, Day 14 postinfection). In contrast, such effect was not observed in the groups preincubated with IgG2 from immune or control sera (Figs. 7A, 7B, 7C). Molecular weight of T. cruzi Ea 4.5 recognized by mouse ZgGl fraction. To define the molecular weight (MW) of T. cruzi Ea 4.5 recognized by IgGl fraction from immunized mice, SDS-PAGE using Ea 4.5 previously separated by IEF was performed, followed by Western blotting. As can be
Parasites
r
25 --
x lO+/ml
I
1
Days post infection
FIG. 5. Effect of treatment of immune and control sera at 56°C for 2 hr. Parasitemia of mice infected with lo3 Tp preincubated with immune- and control-treated sera on Days 10 and 12 postinfection. n , Control; H, immune.
AND
CONTROL
OF
141
PARASITEMIA
x IO-5/ml $0 Parasites --------------
------
------,
I-
!
Dayspost infection FIG. 6. Effect of absorption of immune sera with the glucidic moeity of Ea 4.5. Parasitemia of mice infected with lo3 Tp preincubated with control (W), immune (H), and absorbed immune (Cl) sera on Days 12 and 14 postinfection.
seen in Fig. 8, the IgGl fraction revealed two antigenic bands of MW between 50 and 55 kDa. DISCUSSION
We have previously demonstrated that circulating antigens released by T. cruzi share epitopes with parasite cell surface (Gruppi et al. 1989; Cerban et al. 1991). On the other hand, some experimental evidence shows that membrane antigens can induce a protective immune response (Scott and Snary 1979; Champsi and McMahon-Pratt 1988; Norris et al. 1989). Mouse immunization with Ea 4.5 and Bp as adjuvant produced resistance to T. cruzi infection as revealed by a significantly lower parasitemia and a longer survival of mice immunized compared to control animals (CerbBn et al. 1991). Since the effectiveness of humoral immunity against parasites may depend on the presence of antigen epitopes which bind antibody isotypes capable of mediating protective reactions (Vignali et al. 1989), in this paper we studied the involvement of anti-Ea 4.5 antibody isotypes. Mouse immunization with Ea 4.5 and Bp as adjuvant induced specific IgG with predominance of IgGl. The IgG2 isotype could be
142
CERRAN
, GRUPPI
, AND
VOTTERO-CIMA
looParasitssx 10~%ml
--T--l
b
,,J%fss
x 10-5/ml
--__ I
60.
Days post infection
IL Days pcstinfection
‘-
FIG. 7. Parasitemia of mice infected with 10’ Tp preincubated (A) without sera or with immune and control sera, (B) with immune and control IgGl, (C) with immune and control IgG2, on Days 12 and 14 postinfection. n , Control sera; H, immune sera; q , without sera.
detected only by using EPI as antigen in the ELISA test. In addition, ITH was detected, indicating the presence of reaginic antibodies (IgE and/or IgGl) specific for Ea 4.5. Some authors working with sera from mice chronically infected with Tp of T. cruzi (Takehara ef al. 1981; Brodskyn et al. 1988) or with dead EPI plus complete Freund’s adjuvant (Scott and Goss-Sampson 1984) demonstrated a predominance of specific IgG2 antibodies. In this work the specific IgGl isotype was mainly elicited by K
55 I-) 50-t
FIG. 8. SDS-PAGE of Ea 4.5 followed by Western blotting using immune igG1 (line 1) and control IgGl fraction (line 2). The arrows indicate the position of the bands. The MW of markers from top to bottom are: 94, 67,43, 30, 20.1, and 14.4 kilodaltons &Da).
using a soluble antigen released by T. cruzi (Ea 4.5), Bp as adjuvant, and the immunization schedule already indicated. It is known that humoral immune response to certain antigens is often biased towards the production of particular immunoglobulin isotype (Larsson et al. 1975; Perlmutter et aZ. 1978; Stevens et al. 1983; Callard and Turner 1990). Moreover, the addition of the adjuvant may also contribute to this (Monroy et al. 1989; Karagouni and HadjipetrouKouronakis 1990). Ea 4.5-Bp might result in activation of effector helper cells which would preferentially act on Ea 4.5~specific B cells that have already switched with surface IgGl. This isotype seems to be involved in the parasitemia control of mice challenged with Tp after immunization. The results obtained with treatment of Tp with anti-Ea 4.5 sera indicate that the humoral immunity against Ea 4.5 may play a role in the control of parasitemia in the mice infected postimmunization. This activity was resistant at 56°C suggesting that, in the immunization schedule assayed, the IgGl isotype is the main antibody involved since the other isotypes that may be involved (IgE and IgG2a) are sensitive at this temperature (Damonneville et al. 1986; Weschsler and Kongshavn 1986). In addition, experiments carried out with IgGl and IgG2 purified from immune sera showed
ANTI-T.
Ct74Zi
EXOANTIGENS
AND
that only IgGl isotype was able to decrease the levels of parasitemia in this experimental system. These findings are in agreement with Brodskyn et al. (1989), who obtained some evidences of the ability of IgGl to perform the clearance of parasite bloodstream forms. Furthermore, the ability of parasitemia control of IgGl can now be added to that of IgG2 (Takehara et al. 1981; Scott and GossSampson 1984; Brodskyn et al. 1988) induced during the infection because the parasite can have epitopes that are absent in the Ea 4.5 and which may be able to induce the IgG2 isotype. Even though IgG2 was considered the protector antibody according to the tindings of Takehara et al. (1981) and Brodskyn et al. (1988), it was recently demonstrated that the low-affinity receptor (FcyRII), to which IgGl binds better than IgG2a, becomes a high-affinity receptor by the effect of proteases (van de Winkel et al. 1989), particularly abundant in inflamation sites. The proteases can be released from macrophages after interaction with immune complexes (Cardella et al. 1974) and activated T cells could be another source of proteolytic enzymes (Fruth et al. 1987). Thus, IgGl could efficiently participate in the antibodydependent cellular cytotoxicity or phagocytosis through a receptor different from that of IgG2a and may contribute to the defense mechanisms of the host infected with T. cruzi. Studies about the specificity of the IgGl anti-Ea 4.5 revealed the binding of two components of MW between 50 and 55 kDa. Moreover, it was recently reported that the most relevant epitopes of Ea 4.5 are carbohydrates (Cerban et al. 1991), so we assayed the involvement of antibodies against carbohydrates from Ea 4.5 in the clearance of T. cruzi. It was seen that this capacity was diminished by absorption of immune sera with the glucidic fraction of Ea 4.5. This finding confirms the relevant
CONTROL
OF
PARASITEMIA
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role played by the glucidic epitopes of Ea 4.5, showing that antibodies against these epitopes are important in controlling parasitemia. Further studies will aim to explain how the Ea 4.5 carbohydrate-specific IgGl antibodies interact to eliminate the parasites from T. cruzi-infected mice. ACKNOWLEDGMENTS
The authors are grateful to Dr. Horatio Serra for providing protein A-Sepharose CL-4B and to The Instituto National de Microbiologia “Carlos Malbran” (Buenos Aires) for providing Bordetella pertussis. This work was supported by The Consejo de Investigaciones CientiIicas y Tecnologicas de la Provincia de Cordoba, (CONICOR), Argentina. F. C. thanks CONICOR and A. G. thanks The Consejo National de Investigaciones Cientiticas y Tecnicas (CONICET), Argentina for the fellowships granted. REFERENCES
F. G. 1976. Immunology of Chagas’ disease. I. Circulating antigen in mice experimentally infected with Trypanosoma cruzi. Revista do Institute de Medicina Tropical 18, 433-439. ARAUJO, F. G., CHIARI, E., AND DIAS, J. C. P. 1981. Demonstration of Trypanosoma cruzi antigen in serum from patients with Chagas’ disease. Lancet ARAUJO,
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