Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: immunoprotection against metacyclic trypomastigotes obtained by immunization of mice with an affinity-purified antigen

Molecular and Biochemical Parasitology, 39 (1990) 117-126 Elsevier

117

MOLBIO 01286

Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: immunoprotection against metacyclic trypomastigotes obtained by immunization of mice with an affinitypurified antigen Andr6s Mariano Ruiz, M6nica Esteva, Elisabet Subias, Mirta Moreno, Ada Rosenstein de Campanini, Elsa Velazquez and Elsa Leonor Segura lnstituto Nacional de Diagn6stico e lnvestigaci6n de la Enfermedad de Chagas 'Dr. Mario Fatala Chaben', Buenos Aires, Argentina (Received 20 February 1989; accepted 12 October 1989)

Subcellular fractions of Trypanosoma cruzi (epimastigotes) were assayed in their capacity to induce protective or aggressive effects in experimental animals. The flagellar fraction showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were prepared from mice immunized with this fraction. One of them, FCH-F8-4, was able to neutralize the infectivity of bloodstream trypomastigotes, to produce complement-mediated lysis on cell culture-derived trypomastigotes and to recognize the surface of trypomastigotes and epimastigotes by immunofluorescence. FCH-F8-4 reacted in Western blotting with several epimastigote proteins ranging from 50 to 150 kDa, showing a more intense reactivity with 4 bands while it reacted with two molecules on trypomastigote preparations (15 and 48 kDa). Purified antibody was coupled to CNBr-activated Sepharose and used to purify antigens from epimastigote extracts. These antigens were used to immunize BALB/c mice in the presence of Bordetella pertussis as adjuvant. Animals were protected against a challenge with 103 metacyclic forms of T. cruzi (Tulahu6n strain). Only 40% of immunized mice presented detectable parasites in blood after challenge. Parasitemia decreased 90% in relation to controls in those animals. Survival of immunized mice was 100% in all immunoprotection experiments. These results suggest that the epitope recognized by FCH-F8-4 present in the purified antigens keeps the protective characteristics of flagellar fraction and could be a candidate for the development of a vaccine against T. cruzi infection. Key words: Trypanosoma cruzi antigens; Monoclonal antibody; Experimental immunoprotection

Introduction

of Latin America [1]. The etiological agent Trypanosoma cruzi has a complex life cycle in which

The American trypanosomiasis, Chagas' disease, is widely spread in Central and South America, affecting 12-20 million people out of a population of 65 million living in endemic areas

four stages of the parasite are involved, the epimastigotes and metacyclic trypomastigotes in the insect vector, and the bloodstream trypomastigotes and amastigotes (intracellular forms) in the mammalian host [2]. Vectorial transmission is the main way by which T. cruzi is distributed. Triatomine control is mainly performed by insecticide spraying in houses in endemic areas. These methods have shown to be efficient, mainly due to the participation of the community [3]; however, other preventive methodologies should be developed, such as a vaccine or effective therapeutic treatments. Previous reports have shown that protective

Correspondence address: A.M. Ruiz, Instituto Nacional de Diagn6stico e Investigaci6n de la Enfermedad de Chagas 'Dr. Mario Fatala Chaben', Paseo Col6n 568, 1063 Buenos Aires, Argentina. Abbreviations: IFA, immunofluorescence assay; PMSF, phenylmethylsulfonyl fluoride; TLCK, tosyl lysylchloromethylketone; DAB, 3,3' diaminobenzidine; OPD, o-phenylenediamine-dihydrochloride; aRSV, anti-respiratory syncitial virus monoclonal antibody.

0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

118 immunity against the challenge with infective forms of T. cruzi can be induced using different immunogens [4]. In our laboratory we have described experimental immunoprotection in mice using a flagellar fraction [5,6] of epimastigotes of T. cruzi and Bordetella pertussis as adjuvant, enabling the immunized mice to decrease mortality, parasitemia and histopathology after challenge [7]. Three monoclonal antibodies obtained from a cell fusion made with spleen cells from mice submitted to that immunization scheme [6], showed in vitro complement-mediated lytic (CML) activity against trypomastigotes and were able to confer passive immunoprotection to mice [8]. We show here the immunoprotective activity of another monoclonal antibody obtained from the same fusion and its use to purify antigens which could induce protection in mice against infective forms of T. cruzi. Materials and Methods

Parasites. T. cruzi, Tulahutn strain, Tul2 stock [9] was used in all experiments reported here. Cultured trypomastigotes were obtained from cell cultures [10] and epimastigotes from biphasic medium [11]. Metacyclic trypomastigotes were obtained from the feces of Triatoma infestans bugs which were fed on infected mouse blood [7]; promastigotes of Leishmania mexicana mexicana were obtained from axenic medium [12]. Immunization of animals and cell fusion. The flagellar fraction was obtained as described previously by differential centrifugation of the epimastigotes and purification on a sucrose density gradient [5]. Animals were injected intraperitoneally with lyophilized flagellar fraction (200 p~g per mouse) and B. pertussis as adjuvant (1.25 × 109 O.U.). B. pertussis strain 10536 was obtained from culture, inactivated and controlled, as when incorporated into DTP triple vaccine for children [7]. The third dose was administered without adjuvant by the same route. Cell fusion was performed as previously described [8]. Characterization of monoclonal antibodies. The antibody class was determined by double diffusion in agar using concentrated supernatants re-

acting with antisera specific for immunoglobulin class (Nordic Immunology, Tilburg).

Purification of monoclonal antibodies. Ascites was induced by intraperitoneal inoculation of hybrid cells into BALB/c mice previously injected with pristane. Antibody purification was carried out by using a protein A-Sepharose 4B column (Sigma Chemical Co., St. Louis, MO, U.S.A.). 15 Ixg of the purified preparation were subjected to electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) [14] to check the purity of the antibody. 25 txg of purified antibody was labeled to a specific activity of 2 x 107 cpm (txg protein) -~ with Na125I (Comisi6n Nacional de Energia At6mica, Buenos Aires, Argentina) using the iodogen procedure (Pierce Chemical Co., Rockford, IL). Free 125I was removed using a Sephadex G-25 OS 2A column (Isolab Inc, Akron, OH, U.S.A.) followed by extensive dialysis against phosphatebuffered saline (PBS). Western blotting and determination of the molecular weight of antigen recognized by the monoclonal antibody. 105 culture-derived trypomastigotes or epimastigotes, in the presence of proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and tosyl lysylchloromethylketone (TLCK) with a final concentration of 1 mM per lane, were subjected to SDS-PAGE (10%), and proteins were transferred to nitrocellulose [15]. After saturation with 5% milk (Molico Nestit, Buenos Aires, Argentina), cellulose strips were incubated with 100 I~g ml-~ of monoclonal antibody for 1 h at room temperature (RT). They were washed three times with 0.01% Tween 20 and incubated with (1:500) peroxidase-conjugated goat anti-mouse IgG for 1 h at RT. After this incubation, strips were washed and immersed in 0.05% 3,3'-diaminobenzidine (DAB) (Sigma) 0.1% 1-I202. The reaction was stopped by washing strips with distilled water. Coupling of the monoclonal antibody to CNBractivated Sepharose 4B and purification of antigens from T. cruzi epimastigote extracts. 40 mg of purified antibody (4 mg m1-1) was dialyzed overnight against 1 M NaHCO3, 150 mM NaC1, pH

119 8.0 (coupling buffer), and coupled to CNBr-activated Sepharose 4B according to the method suggested by the manufacturer (Pharmacia Fine Chemicals, Uppsala, Sweden). The beads were equilibrated with Tris-buffered saline (TBS) and stored at 4°C pending antigen purifications. 2 g wet mass of Tulahu6n strain epimastigotes were suspended in TBS/0.1% NP 40/1 mM PMSF/1 mM TLCK. The suspension was centrifuged at 15 000 × g and the supernatant added to the top of the monoclonal antibody-Sepharose column and passed twice. After washing with TBS until A280=0, the bound antigens were eluted with glycine-HC1 buffer, pH 2.4. 1-ml fractions were collected on 1.6 ml of TBS. Tubes containing proteins were pooled and dialyzed, and several aliquots were subjected to SDS-PAGE (10%). Protein concentration was determinated by the method of Lowry [16]. Preparations were kept at 70°C awaiting lyophilization and use in immunoprotection experiments [7]. -

Indirect immunofluorescence assays. Immunofluorescence was performed using multiple-well slides containing trypomastigotes or epimastigotes fixed in 0.1% glutaraldehyde. After reaction with the monoclonal antibodies or immunosera, the washed slides were incubated with fluorescein isothiocyanate-conjugated goat immunoglobulin anti-mouse IgG (Byosis, Compi~gne, France). Immunoenzymatic assay (ELISA). 96-well microtiter plates coated with 5 × 103 trypomastigotes or epimastigotes per well were saturated with 1% normal goat serum, 1% BSA. Additional plates, without antigens, coated with the same solution, were simultaneously used as control. 25 i~1 of varying concentrations of monoclonal antibodies or serum dilutions were added to each "well and incubated for 1 h at RT. They were washed with 0.01% Tween 20 and were incubated with 30 txl peroxidase-conjugated goat anti-mouse IgG for 1 h at RT. After washing, 200 Ixl of a solution of 0.05% o-phenylenediamine-dihydrochloride (OPD; Sigma) 0.1% H20 2 was added to each well. Plates were read at 490 nan in a ELISA reader after stopping the reaction with 2.5 M HCI.

Neutralization of T. cruzi infectivity. Parasites were obtained by infecting irradiated BALB/c mice [8]. Suspensions of trypomastigotes were incubated with different amounts of monoclonal antibodies (anti-T. cruzi or unrelated) for 60 min at 37°C. After the incubation period, parasites were inoculated intraperitoneally into two different groups of BALB/c mice (500 parasites per 500 ~1 per mouse). Parasitemia and survival were recorded until day 40 post-infection. Complement-mediated immunolysis on trypomastigotes. Complement-mediated immunolysis was carried out as previously described [8]. 100 Ixl of cell culture trypomastigotes (2.5 x 106 parasites m1-1) were incubated with the same volume of monoclonal antibodies (anti-T. cruzi or unrelated) (1 mg ml-1). Immunoprotection experiments. BALB/c mice were injected with antigen purified by affinity chromatography as described above (100 ~g per 500 ~1 per mouse) and B. pertussis (1.25 × 109 O.U. per mouse) as adjuvant. Another group was injected with the adjuvant alone as control and an additional group was injected with BSA. All three groups were challenged with 103 metacyclic parasites per mouse. Parasitemia and survival were studied until day 40 post-infection. Sera from the three groups were taken after the second dose of the immunogen, to test for the presence of antibodies reacting with trypomastigotes in ELISA. Competitive immunoradiometric assay to detect anti-T, cruzi antibodies in human sera. A competitive assay was done to detect the presence of antibodies reacting with FCH-F8-4 purified antigen in human sera. In this assay, 25 sera reacting with epimastigotes antigens by three different serological techniques (IFA, complement fixation reaction and hemagglutination) were used, 25 sera from normal individuals, and 3 sera from patients with leishmaniasis from the north of Argentina. (Sera were kindly provided, by Dr. Estela Cura, INDIECH, Buenos Aires, Argentina). 96-well flexible microtiter plates were coated with 100 I~g m1-1 purified antigen (50 ixl well -1) and left overnight at RT. Plates were saturated with 1% BSA for 2 h at RT. 50 Ixl of a 1:400 di-

120

lution of each serum were mixed with the same volume of Na125I-labeled antibody (30 x 1@ cpm) and 25 ~1 of that solution was added to each well of the antigen-coated plates and incubated for 2 h. Then, plates were washed, dried and cut to be counted in a gamma counter (Picker Compac 120, Picker Corporation, U.S.A.). Unlabeled monoclonal antibody was used as control for competition by the same epitope. Results

As previously described [8], 19 positive hybridomas were obtained in this cell fusion. We report only one of them in this study. This hybri-

kDa

[]

•

•

0

94.0 ~ . . 67.0

30.0~_~

i!i!i!;iii!iiii

20.0 := 14.4 ~ A

B

Fig. 1. (A) Reactivity of FCH-F8-4 on trypomastigotes and epimastigotes by Western blotting. 106 trypomastigotes or epimastigotes (10 5 parasites/lane) were subjected to electrophoresis on SDS-polyacrylamide gels (10%) and proteins were electrotransferred to nitrocellulose sheets. Reaction of monoclonal antibodies (FCH-F8-4 or aRSV) on nitrocellulose strips were assayed as described. Reactivity of FCH-F8-4 on epimastigote (a) and trypomastigote ( I ) strips, aRSV monoclonal antibody did not recognize anything on both parasite forms. (B) SDS-PAGE and Western blotting of antigens purified by affinity chromatography using FCH-F8-4 monoclonal antibody. Purified monoclonal antibody coupled to CNBr-Sepharose as described in Materials and Methods was used to purify antigens from a epimastigote preparation. Tubes containing the purified proteins were pooled, dialyzed and subjected to SDS-PAGE (10%), and the gels were stained with Coomassie Blue (o) or electrotransferred onto nitrocellulose sheets and developed with Na125I-labeled FCH-F8-4 monoclonal antibody (e).

doma was cloned by limiting dilution and its secreted antibody characterized as described in Materials and Methods. The immunoglobulin isotype of this monoclonal antibody (FCH-F8-4) is an IgG> An unrelated anti-respiratory syncitial virus monoclonal antibody (aRSV), kindly given by Dr. Mercedes Weissenbacher, University of Buenos Aires, was used as negative control in all experiments reported here. Antibodies were purified from ascitic fluids produced in primed BALB/c mice after the inoculation of both hybridomas. FCH-F8-4 monoclonal antibody was able to recognize the surface of cultured trypomastigotes and epimastigotes as detected by IFA. Reactivity with both forms was also found in ELISA (data not shown). When this antibody was assayed by means of Western blotting, it was noticed that it reacted with several proteins of epimastigotes, ranging from 50 to 150 kDa, showing a more intense reactivity with 4 bands, while it reacted with two molecules on trypomastigote preparations (15 and 48 kDa) (Fig. 1). There was no reactivity with L. mexicana mexicana in this assay. Bloodstream trypomastigotes from previously irradiated BALB/c mice were partially neutralized in their capacity to infect mice after the incubation with FCH-F8-4. Survival of mice which received parasites plus a different amount of FCH-F8-4 was 40-80% higher than in those receiving parasites plus aRSV. A delay in mortality of mice with respect to control animals was observed in animals receiving parasites plus FCH-F84. Neutralization of infectivity was more effective in groups of mice which received the highest dose of antibody vs. the same amount of parasites (Table I). Six different experiments showed FCH-F8-4 to have complement-mediated lytic activity on cellculture-derived trypomastigotes. Lysis of parasites incubated with FCH-F8-4 ranged between 40 and 100% (60.8 +-- 17.1%) through the six experiments, while those with aRSV did not show more than 20% (6.3 +-- 10.9%) (Table I). FCH-F8-4 monoclonal antibody was used to purify antigens from epimastigotes, as described in Materials and Methods. When the antigens were analyzed by SDS-PAGE (10%) and stained with Coomassie Blue, several bands were found.

121 TABLE I Neutralization of infectivity in BALB/c mice and complement-mediated lysis of trypomastigotes by FCH-F8-4 monoclonal antibody Monoclonal antibody

% Survival after inoculation of parasites+Ab" (day of prepatency -+ SD) 150b 200 300 400 600

anti-RSV

0 (21.4-+5.8)

0 (19.2-+3.4)

20 (17.6-+0.8)

FCH-F8-4

60 (27.0-+3.5)

60 (29.5-+5.5)

60 (27.5-+6.0)

20 (17.3-+3.0) 100 -

% CML of culture trypomastigotes -+ S D d

20 (21.5-+2.5)

6.3-+ 10.9

80 (38.0-+1.2)

60.8-+ 17.1

aSame results found in three different experiments. t'I'otal amount of antibody added per mouse in ~g. cSerum dilution. dResults are the average of 6 experiments with the same schedule.

T h e s e a n t i g e n s w e r e e l e c t r o t r a n s f e r r e d o n t o nit r o c e l l u l o s e s h e e t s a n d d e v e l o p e d with Na125I lab e l e d F C H - F 8 - 4 m o n o c l o n a l a n t i b o d y , showing that all m o l e c u l e s w e r e r e c o g n i z e d on all p r e p a r a t i o n s (Fig. 1). T h e c o m p e t i t i v e assay was a b l e to d e t e c t antib o d i e s r e a c t i n g with F C H - F 8 - 4 purified antigen. O u t o f t h e 25 p o s i t i v e s a m p l e s by t h e t h r e e techniques, 18 s h o w e d 7 0 % i n h i b i t i o n in t h e c o m p e -

tition b i n d i n g test. Six h a d a 60% i n h i b i t i o n , a n d 1 did n o t inhibit the r e a c t i o n . T h e s e r a f r o m n o r m a l i n d i v i d u a l s o r f r o m p a t i e n t s with l e i s h m a n iasis d i d n o t show i n h i b i t i o n (Fig. 2). In t h e imm u n o p r o t e c t i o n e x p e r i m e n t s , t h e activity o f the purified antigen plus adjuvant was c o m p a r e d with results o b t a i n e d in mice i m m u n i z e d with a d j u v a n t alone. A t the e n d o f the s e c o n d d o s e o f imm u n o g e n (15 days after the first), mice i n j e c t e d

22000

0

20000

0

18000

zl A

0

16000 nO E :3 0 ..,Q

0 0

rn o O~

14000

0

%

0

12000 10000- •

o i O 0

0

0

•

0

E M'

6000 4000

•

-

xxx

ooor, -.. 0 0

I 10

I 20

I

I

I

30

40

50

] 6O

Sero t e s t e d

Fig. 2. Competitive immunoradiometric assay by the FCH-F8-4 purified antigens using human sera. 50 Ixl of 1:400 human sera were incubated for 30 min with 50 ~.l (30 × 103 cpm) of Na125I-labeledFCH-F8-4 monoclonai antibody. 25 I~l of this solution was added to each well of plates coated with purified antigen, as described in Materials and Methods. After washing, plates were counted. Results are shown as cpm bound when labeled monoclonal antibody was incubated with: 0, sera from seroiogically reactive chagasic patients; o, sera from normal individuals; A, A, sera from patients with leishmaniasis form the North of Argentina; x, unlabeled FCIq-F8-4 monoclonal antibody. Population of sera inhibiting the reaction were significantly different from controls (sera from normal individuals or patient with leishmaniasis), P < 0.001, t-test.

122

B IFA titers 1:8 O 1:8 • 1:16 O 1:16

sponse of mice ranged from 0.2 to 0.65 at 1:1600 (v/v) by ELISA, while that in IFA the reactivity of those sera ranged from 1:8 to 1:32 (v/v), showing a correlation between both techniques. Animals were protected against a challenge with 103 metacyclic forms/mouse. Only 40% of immunized mice presented parasites in blood. Parasitemia decreased 90% with respect to control in those animals. Survival of immunized mice was 100% in both experiments (Table II).

•

•

1:32

A Average of five serQ injected 1.2 - with B, pertussis (IFA:neg)

1.C ~~A 0,6 # 0.4 ul ~3

<

•

Discussion

Q2 O.C 100

200

400

800

1 6 0 0 3200

Reciprocal of serum dilution Fig. 3. (A) Reactivity of sera from mice immunized with FCH-F8-4 affinity-purified antigen, on trypomastigotes by ELISA. 96-well microtiter plates were coated with culture derived trypomastigotes and saturated as described in Materials and Methods. 25 Ixl of different dilutions of mouse sera were added to each well and incubated for 2 h at RT. Five sera from mice injected with B. pertuss& alone were included as negative control. Later the incubation plates were treated as previously described. (B) Titration of sera from mice immunized with FCH-F8-4 affinity-purified antigen by indirect immunofluorescence assay (IFA). Glutaraldehyde-fixed culture trypomastigotes on 24-well slides were incubated with 10 ixl of different dilutions of sera for 30 min at 37°C. After washing with PBS, slides were incubated with fluorescein isothiocyanate-conjugated goat anti mouse immunoglobulin (IgG). B. pertussis injected mouse sera were used as controls.

with FCH-F8-4 affinity-purified antigen, showed an antibody response against trypomastigotes as measured by ELISA (Fig. 3). The individual re-

The monoclonal antibody FCH-F8-4 studied in this work was prepared from mice immunized with the flagellar fraction of epimastigotes of T. cruzi (Tulahu6n strain). This fraction was able to confer protection to immunized animals against a challenge with infecting forms [5,6]. This fact indicates that flagellar fraction contains antigens which are also present in those trypomastigotes. The results shown here confirm this fact since FCH-F8-4 recognizes both forms of the parasite. The multiple reactivity with epimastigotes preparations would indicate that a 'public' epitope is present in different molecules of the parasite; perhaps some of them could be intracellular proteins or precursors. On trypomastigotes, this epitopes is also present, although in fewer proteins. Reactivity on trypomastigotes was also confirmed by immunofluorescence, neutralization of their infectivity in BALB/c mice at different concentrations of antibody, and complement-me-

T A B L E II Survival and parasitemia of mice challenged with Trypanosoma cruzi after immunization with FCH-F8-4 affinity purified antigen Expt.

Immunogen

Survival (%) after challenge ~ 10 20 30

40

Mice infected (%)

Bp BSA Bp+Ag

100 100 100

60 60 100

60 60 100

60 60 100

100 100 40

1.35±0.21 (22) 1.52±0.32 (22) 0.11±0.10 (23) c

Bp BSA Bp+Ag

100 100 100

70 60 100

60 50 100

55 50 100

100 100 40

0.95±0.22 (25) 1.24±0.50 (24) 0.15±0.08 (25) c

~Days post-infection. bData at the peak of parasitemia. ~Statistically significant difference from Bp and BSA immunized mice. P<0.001, t-test.

Parasitemia b (ml t x 10 -6 ± SD) (day)

123 diated lytic activity. Complement-mediated lysis of bloodstream trypomastigotes was also reported with another monoclonal antibody (FCHF8-1) obtained from the same cell fusion [8] showing that antigens eliciting protective activity are present in the flagellar fraction. Protective and lytic activity have been reported as an exclusive property of antibodies from chronically infected mice [17]. In a recent review, Brener disregards any vaccine constructed with immunizing antigens [18]. We report this activity among the biological characteristics of this monoclonal antibody obtained from mice immunized with a fraction free of infecting parasites. Other reports have shown lytic activity in antisera from immunized mice [19]. In addition, we also report here the neutralizing activity of FCH-F8-4 on trypomastigotes. The role of antibodies in protective mechanisms against T. cruzi infection has been intensively studied. Agglutination and lysis of trypomastigotes were described as part of that action [17]. On the other hand, reproducible results were obtained by inoculating mice with trypomastigotes incubated with immunosera. A decrease in the lethal activity of the parasite was reported compared to control groups which received trypomastigotes incubated with normal serum. This action was observed even when the immunosera did not produce lysis on the parasite [20]. The presence of neutralizing antibodies like FCH-F84 might be involved in this modification of the infectivity in the mouse immune response. The family of monoclonal antibodies described by our laboratory has shown several properties, and different specificities to T. cruzi. They were selected for the purification of antigens, according to their characteristics. Purification of antigens from trypomastigotes with FCH-F8-1 monoclonal antibody using affinity chromatography methods was practically impossible. This monoclonal antibody showed a very low affinity for the parasite (J. Bua, personal communication). Instead, we were able to purify antigens using epimastigote extracts and FCH-F8-4. The antigens obtained in these purifications contain all the molecules which are recognized by the monoclonal antibody on epimastigote preparation; this was confirmed by immunoblotting of the purified

product and developing with labeled monoclonal antibody. Coomassie Blue staining of the gels revealed fewer bands in the purified preparation, due to its lower sensitivity compared to the blotting. To discover whether these antigens kept the protective properties present in the original preparations, they were injected into different groups of BALB/c mice in the presence of B. pertussis as adjuvant. Immunized mice were able to evoke an immune response which conferred protection against challenge with metacyclic forms of Tulahu6n strain of T. cruzi, in terms of survival and parasitemia. This immunization could not protect them against the presence of parasitemia, but only 40% of animals had parasites detected microscopically, after challenge, while in the control groups (animals immunized with B. pertussis alone) all mice presented high parasitemias. We still do not know if the chronic pathology can be prevented, as seen when mice are immunized with fiagellar fraction. This monoclonal antibody and its purified antigen were used simultaneously to detect antibodies reacting with T. cruzi in human sera, evaluating its diagnostic capacity. Several sera were tested in a competitive assay. On 25 samples reactive by three techniques, only one serum did not inhibit the reaction. Surface antigens of T. cruzi have exhaustively been studied, using immunoprecipitation techniques [21-24], and their reactivity with monoclonal antibodies [25-28] or lectins [28--30]. A significant number of molecules was detected in epi-, trypo- or amastigotes, some of them present in only one stage. The biological characteristics of those molecules were analyzed by several methods, with different results. A 90-kDa glycoprotein present in the three forms of the parasite (Y strain) was purified and used in immunoprotection experiments with good results when tested against the acute experimental disease [29]. This glycoprotein was also tested on its adjuvant requirements, to improve the protection, showing the role of the cellular immune response in the immunized animals [31]. In a recent report, we also showed that the immunization with flagellar fraction and B. pertussis can induce cellular mechanisms critically

124 involved in the protection of mice [32]. A similar molecule was also reported, although present only on bloodstream trypomastigotes [33], and a 100-kDa protein not detectable on Y and C1 strains was described as the main surface antigen on G metacyclic trypomastigotes [34]. A trypomastigote stage-specific 85-kDa glycoprotein was described as responsible for the internalization process of T. cruzi into mammalian cells [35]. Ouaissi et al. identified a similar protein as a fibronectin receptor [36]. In our laboratory, we described another 85-kDa molecule detected using FCH-F8-1 monoclonal antibody. Although this protein was not purified, the monoclonal antibody showed protective characteristics [8]. The gene coding for an 85-kDa surface antigen of T. c r u z i was cloned, identifying a nonapeptide repeat [37]. The synthetic peptide is not recognized by FCH-F8-1 or FCH-F8"4 monoclonal antibodies on E L I S A , with the peptide coated on 96-well microtiter plates (J. Bua, personal communication). A 72-kDa specific protein from epimastigotes [24] was able to induce protection in mice against a challenge with metacyclic forms, while they were not protected against blood parasites [38]. The 72kDa glycoprotein was reported to be a membrane acceptor site for C3 on epimastigotes [39]. Antigens purified by FCH-F8-4 were also able to confer protection against metacyclic trypomastigotes. We do not know if mice can be protected against bloodstream parasites, although the antibody used to purify them could neutralize and lyse those infective forms.

All these reports show that a large group of proteins are involved in the interaction mechanisms with the host cells. Some of them have protective properties against infection with T. cruzi. However, purified and defined antigens were used as immunogens in only few assays. Purification of large amounts of antigens from the different parasite stages has been difficult, and only genetically engineered or synthetic vaccines could really be contemplated. The results presented in this paper show the possibility of obtaining protective antigens from this family of antibodies prepared from mice immunized with flagellar fraction of T. cruzi epimastigotes. Molecular biological techniques could lead to the obtainment of optimal candidates for the development of a vaccine and/or diagnostic tool for T. c r u z i infection.

Acknowledgements We wish to thank Drs. Juan Jos6 Cazzulo and Mirta Carlomagno for the critical revision of this manuscript. This investigation received financial support from the U N D P / W o r l d B a n k / W H O Special Programme for Research and Training in Tropical Diseases and the Ministerio de Salud y Accion Social and the Secretaria de Estado de Ciencia y T6cnica of Argentina. E.L.S. is a member of the Carrera del Investigador Cientifico and E.V. of the Carrera de Personal de A p o y o a la Investigaci6n, both of the Consejo Nacional de Investigaciones Cientificas y T6cnicas of Argentina.

References 1 Maekelt, G.A. (1983) La Epidemiologia de la Enfermedad de Chagas en relaci6n con el ecosistema domiciliario. Intersciencia 8,353-366. 2 Brener, Z. (1973) Biology de Trypanosoma cruzi. Annu. Rev. Microbiol. 27,347-383. 3 Wisnivesky-Colli, C., Paulone, I., Perez, A., Chuit, R., Gualteri, J., Solarz, N., Smith, A. and Segura, E.L. (1987) A new tool for continuous detection of the presence of Triatomine bugs, vectors of Chagas' disease, in rural households. Medicina (Buenos Aires) 47, 45-50. 4 Scott, M.T. and Snary, D. (1982) American Trypanosomiasis (Chagas' disease) In: Immunology of Parasitic Infections, (Cohen, S. and Warren, K., Eds.), 2nd. ed., pp. 261-298. BlackweU, Oxford.

5 Segura, E.L., Vazquez, C., Bronzina, A., Campos, J.M., Cerisola, J.A. and Gonzalez Cappa, S.M. (1977) Antigens of the subcellular fractions of Trypanosoma cruzi, II. FlageUar and membrane fraction. J. Protozool. 24, 540-543. 6 Ruiz, A.M., Esteva, M., Cabeza Meckert, P., Laguens, R.P. and Segura, E.L. (1985) Protective and pathology induced by inoculation of mice with different subcellular fractions of Trypanosoma cruzi. Acta Trop. 42, 299-309. 7 Ruiz, A.M., Esteva, M., Riarte, A., Subias, E. and Segura, E.L. (1986) Immunoprotection of mice against Trypanosoma cruzi with a lyophilizedflagellar fraction of the parasite plus adjuvant. Immunoi. Lett. 12, 1-4. 8 Segura, E.L., Bua, J., Rosenstein de Campanini, A., Subias, E., Esteva, M., Moreno, M. and Ruiz, A.M. (1986)

125 Monoclonal antibodies against the flagellar fraction of epimastigotes of Trypanosoma cruzi: complement-mediated activity against trypomastigotes and passive immunoprotection in mice. Immunol. Lett. 13, 165-171. 9 Segura, E.L., Subias, E., Esteva, M., Cabeza Meckert, P., Bronzina, A. and Laguens, R.P. (1980) Caracteristicas de infectividad de tres poblaciones de cultivo de Trypanosoma cruzi. Medicina (Buenos Aires) 40, 97-102. 10 Doyle, P.S., Dvorak, J.A. and Engel, J.C. (1984) Trypanosoma cruzi: Quantification and analysis of the infectivity of cloned strains. J. Protozool. 31,280-283. 11 Gerez de Burgos, N.M., Burgos, C., Blanco, A., Paulone, I. and Segura, E.L. (1976) Actividad alfa hidroxiacido dehidrogenasa en Trypanosoma cruzi. Acta Physiol. Latinoamer. 26, 10-17. 12 Camargo, E.P. (1964) Growth and differentiation in Trypanosoma cruzi, I. Origin of metacyclical trypanosomes in liquid media. Rev. Inst. Med. Trop. S. Paulo 6, 93-100. 13 Murphy, M., Rasnack, J. and Disckon, M. (1983) Evaluation of the pertussis components of DTP vaccines. Pediatrics 71,200-204. 14 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. 15 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350--4354. 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 93,265-275. 17 Krettli, A.U. and Brener, Z. (1982) Resistance against Trypanosoma cruzi associated to anti living trypomastigote antibodies. J. Immunol. 128, 2009-2010. 18 Brener, Z. (1986) Why vaccines do not work in Chagas' disease. Parasitol. Today 2, 196-197. 19 Yoshida, N. (1986) Trypomastigote cruzi, recognition of trypomastigote surface antigens by lytic antisera from mice resistant to acute infection. Exp. Parasitol. 61, 184-191. 20 Gonzalez Cappa, S., Katzin, A. and Lajmanovich, S. (1979) Trypanosoma cruzi. Actividad de immunosueros de rat6n sobre el estadio tripomastigote. Medicina 39, 187-192. 21 Nogueira, N., Unkeless, J. and Conn, Z. (1982) Specific glycoprotein antigens on the surface of insect and mammalian cells stages of Trypanosoma cruzi. Proc. Natl. Acad. Sci. USA 79, 1259-1263. 22 Andrews, N.W., Katzin, A. and Colli, W. (1984) Mapping of surface proteins and antigens of Trypanosoma cruzi by two dimensional electrophoresis, a correlation with the cell invasion capacity. Eur. J. Biochem. 140, 599-604. 23 Gonzalez, N.S., Sanchez, D.O., Frasch, A.C. and AIgranati, I. (1984) Surface proteins in different isolates of Trypanosoma cruzi epimastigotes. Mol. Cell. Biochem. 63, 157-164. 24 Snary, D., Ferguson, M.A., Scott, M.T. and Allen, A. (1981) Cell surface antigens of Trypanosoma cruzi, use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein. Mol. Biochem. Parasitol. 3, 343-356.

25 Kirchoff, L.Y., Engel, J.C., Dvorak, J.A. and Sher, A. (1984) Strains and clones of Trypanosoma cruzi differ in their expression of a surface antigen identified by a monoclonal antibody. Mol. Biochem. Parasitol. 11, 81-89. 26 Orozco, O., Afchain, D., Dissous, C., Rodriguez, C., Ovlaque, G., Lemestre, J.L., Loyens, M. and Capron, A. (1984) Different monoclonal antibodies against the component 5 specific for Trypanosoma cruzi. Am. J. Trop. Med. Hyg. 33,560-568. 27 Teixeira, M.M.G. and Yoshida, N. (1986) Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies. Mol. Biochem. Parasitol. 18,271-282. 28 Katzin, A.M. and Colli, W. (1983) Lectin receptors in Trypanosoma cruzi, and N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage. Biochim. Biophys. Acta 727,403-411. 29 Snary, D. and Hudson, L. (1979) Trypanosorna cruzi cell surface proteins, identification of one major glycoprotein. FEBS. Lett. 100, 166-170. 30 Dragon, E.A., Brothers, Y.N., Wrightman, R.A. and Manning, J. (1985) A M r 90000 surface polypeptide of Trypanosoma cruzi as a candidate for a Chagas' disease diagnostic antigen. Mol. Biochem. Parasitol. 16, 213-229. 31 Scott, M.T., Bahr, G., Moddaber, F., Afchain, D. and Chedid, L. (1984) Adjuvant requirements for protective immunization of mice using a Trypanosoma cruzi 90 K cell surface glycoprotein. Int. Archs. Allergy Appl. Immunol. 74, 373-377. 32 Rottenberg, M., Cardoni, R., de Titto, E., Moreno, M. and Segura, E.I. (1988) Trypanosoma cruzi, Immune response in mice immunized with parasite antigens. Exp. Parasitol. 65, 101-108. 33 Nogueira, N., Chaplan, S., Tydings, J.D., Unkeless, J., Cohn, Z. (1981) Trypanosoma cruzi: Surface antigens of Blood and culture forms. J. Exp. Med. 153, 629-639. 34 Yoshida, N. (1983) Surface antigens of metacyclic Trypomastigotes of Trypanosoma cruzi. Infect. Immun. 40, 836-839. 35 Zingales, B., Andrews, N., Kuwajima, Y.Y. and Colli, W. (1982) Cell surface antigens of Trypanosoma cruzi: possible correlation with the interiorization process in mammalian cells. Mol. Biochem. Parasitol. 6, 111-124. 36 Ali Ouaissi, M., Cornette, J. and Capron, A. (1986) Identification and isolation of Trypanosoma cruzi trypomastigote cell surface protein with properties expected of a fibronectin receptor. Mol. Biochem. Parasitol. 19,201-211. 37 Peterson, D., Wrightsman, R. and Manning, J.E. (1986) Cloning of a major surface antigen gene of Trypanosoma cruzi and identification of a nonapeptide repeat. Nature 322,566-568. 38 Snary, D. (1983) Cell surface glycoproteins of Trypanosoma cruzi, protective immunity in mice and antibody levels in human chagasic sera. Trans. R. Soc. Hyg. 77, 126-129. 39 Joiner, K., Heiny, S., Kirchhoff, L. and Sher, A. (1986) GP72, the 72 000 Dalton glycoprotein, is a membrane acceptor site for C3 on Trypanosoma cruzi epimastigotes. J. Exp. Med. 161, 1196--1212.