Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3541-3545, April 1995 Immunology

Autoimmunity in Chagas disease cardiopathy: Biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen EDIECIO CUNHA_NETO*, MARCIA DURANTI*, ARTHUR GRUBERt, BIANCA ZINGALESt, IARA DE MESSIASt, NOEDIR STOLF§, GIovANNI BELLOTTI§, MANOEL E. PATARROYOI, FuLvIo PILLEGGI§, AND JORGE KALIL*II *Laboratory of Transplantation Immunology, §Divisions of Surgical and Clinical Cardiology, Instituto do Coracao, Sao Paulo University Hospital, Faculty of Medicine, and tlnstituto de Quimica, Universidade de Sao Paulo, Sao Paulo, SP, 05403-000, Brazil; tImmunopathology Laboratory, Hospital de Clinicas, University of Parana, Curitiba, Brazil; and Instituto de Inmunologia, Hospital San Juan de Dios, Universidad Nacional de Colombia, Bogota, Colombia Communicated by Jean Dausset, Centre d'Etude du Polymorphisme Humain, Paris, France, October 21, 1994

ABSTRACT Heart tissue destruction in chronic Chagas sensitivity responses toward a tissue-specific heart component disease cardiopathy (CCC) may be caused by autoimmune bearing structural similarities to a given T. cruzi antigen. recognition of heart tissue by a mononuclear cell infiltrate Several investigators found autoantibody and self-reactive decades after Trypanosoma cruzi infection. Indirect evidence T-cell formation in human and experimental T cruzi infection suggests that there is antigenic crossreactivity between T. (5, 6). Crossreactive autoantibodies were mainly directed cruzi and heart tissue. As there is evidence for immune toward ubiquitous and evolutionarily conserved molecules (7, recognition of cardiac myosin in CCC, we searched for a 8). A recent report (9) demonstrated an epitope shared putative myosin-crossreactive T. cruzi antigen. T. cruzi lysate between a recombinant flagellar T. cruzi molecule (Fl 160) and immunoblots were probed with anti-cardiac myosin heavy an undefined 48-kDa neuronal protein. However, Fl 160-nerve chain IgG antibodies (AMA) affinity-purified from CCC or crossreactive antibodies did not correlate to CCC or chronic asymptomatic Chagas disease patient-seropositive sera. A digestive forms of human Chagas disease. Most T. cruzi-host 140/116-kDa doublet was predominantly recognized by AMA tissue crossreactive antigen systems described seemed to lack from CCC sera. Further, recombinant T. cruzi protein B13- clinico-biological significance, as indicated by a lack of target whose native protein is also a 140- and 116-kDa double organ specificity of self-antigen as well as lack of correlation band-was identified by crossreactive AMA. Among 28 sera between antibody detection and different clinical forms of tested in a dot-blot assay, AMA from 100%6 of CCC sera but chronic Chagas disease. only 14% ofthe asymptomatic Chagas disease sera recognized The recognition of myosin by CD4+ T cells in a murine B13 protein (P = 2.3 x 10-6). Sequence homology to B13 model of CCC (10) revealed a prime candidate target autoan- protein was found at positions 8-13 and 1442-1447 of human tigen in CCC. Myosin heavy chain (myosin HC) is the most cardiac myosin heavy chain. Competitive ELISA assays that abundant heart protein-50% of total protein by weight used the correspondent myosin synthetic peptides to inhibit (11)-and is recognized in many situations of heart-specific serum antibody binding to B13 protein identified the heart- autoimmunity, such as rheumatic heart disease (12) and specific AAALDK (1442-1447) sequence of human cardiac murine post-Coxsackie B3 autoimmune cardiomyopathy (13). myosin heavy chain and the homologous AAAGDK B13 se- The presence of antibody against cardiac myosin HC is cor- quence as the respective crossreactive epitopes. The recogni- related to the development of chronic inflammatory cardio- tion ofa heart-specific T. cruzi crossreactive epitope, in strong pathy in T cruzi-infected mice (14). Immunization with cardiac association with the presence of chronic heart lesions, sug- myosin HC induces an aggressive myocarditis (15, 16). These gests the involvement of crossreactivity between cardiac my- reports prompted us to look for anti-cardiac myosin HC osin and B13 in the pathogenesis of CCC. immune reactivity in CCC patients and search for a possible myosin crossreactive T. cruzi antigen. Chagas disease (American trypanosomiasis), caused by the protozoan Trypanosoma cruzi, is endemic in many countries of Latin America, where 16-18 million people may be infected. MATERIALS AND METHODS Chronic Chagas disease cardiopathy (CCC), a dilated cardiom- Patients. CCC patients (severe heart failure, dilated car- yopathy with a T-cell-rich inflammatory infiltrate that often diomyopathy with a positive serology for T. cruzi, and all other leads to a fatal course, develops in 25-30% of infected causes excluded) in waiting list for cardiac transplantation individuals 5-30 yr after primary infection (1). The remaining were subjected to transvenous endomyocardial biopsy. Sample 60-70% chronically T. cruzi-infected individuals either remain collection procedures have been cleared by the Committee of asymptomatic ("indeterminate" patients, ASY) or develop Ethics. Peripheral blood was collected from 24 CCC patients, denervation of parietal smooth muscle in the digestive tract as well as from 14 asymptomatic Chagas disease patient (ASY) (5-10%). individuals (clinically asymptomatic individuals seropositive to The pathogenesis of heart lesions in CCC has been the issue T. cruzi, with a normal electrocardiograph and heart sonog- of much debate. Several lines of evidence-including the from Instituto do Cora-ao and from inability to consistently find parasites in damaged areas (2) and raphy) Parana University the passive transfer of heart lesions by CD4+ T cells from T. Hospital outpatient clinics, as well as from 10 normal volun- cruzi-infected mice (3)-indicated that the inflammatory heart teers. Sera from Duchenne muscle dystrophy patients were lesion could be of an autoimmune nature (4). provided by Mayana Zatz, University of Sao Paulo. His- According to the antigenic mimicry hypothesis, lymphocytes in the heart should recognize and mount delayed-type hyper- Abbreviations: CCC, chronic Chagas disease cardiopathy; ASY, asymptomatic Chagas disease patient; myosin HC, myosin heavy chain; AMA, anti-cardiac myosin HC IgG antibodies; NR, nonrelated pep- The publication costs of this article were defrayed in part by page charge tide; pB13, 19-mer that encompasses all possible linear epitopes within payment. This article must therefore be hereby marked "advertisement" in the 12-mer tandem repeats of B13 protein. accordance with 18 U.S.C. §1734 solely to indicate this fact. IlTo whom reprint requests should be addressed. 3541 Downloaded by guest on September 27, 2021 3542 Immunology: Cunha-Neto et al Proc. Natl. Acad Sci USA 92 (1995) topathological scoring for myocarditis of biopsy samples was anti-human IgG-alkaline phosphatase conjugate (Sigma), and done as described (17). developed with nitro blue tetrazolium/5-bromo-4-chloro-3- Antigens. Purified human cardiac myosin (for ELISA), as indolyl phosphate chromogens. T. cruzi strips were alterna- well as myosin-rich 0.3 M KCl soluble fraction of heart tively incubated with a rabbit antiserum to B13 recombinant homogenate (for immunoblots and immunosorbent prepara- protein, when a goat anti-rabbit IgG-alkaline phosphatase tion), was obtained from normal human heart ventricular conjugate (Sigma) was used. Recombinant B13 protein and tissue of an organ donor as described (18). After SDS/7.5% nonrecombinant ,B-galactosidase were dot-blotted on nitrocel- PAGE (19), the myosin-rich heart lysate was electroblotted to lulose membranes with a BRL 96-well manifold (50 ng per nitrocellulose sheets (20). Cardiac myosin HC-containing ni- dot). AMA of all tested sera were incubated overnight at 4°C trocellulose strips (major 210-kDa protein band) were used as and processed as described for immunoblots. immunosorbents. Tissue culture-derived T. cruzi Y strain ELISA Assays. Anti-myosin antibody assays were done trypomastigotes obtained from infected LLC-MK2 cell line essentially as described (13). In our system, 1:200 dilutions of monolayers (21) were fractionated by SDS/7.5% PAGE in the individual sera were incubated overnight, at 4°C, in triplicates, presence of 2-mercaptoethanol, being subsequently trans- over purified human cardiac myosin as the solid-phase antigen, ferred to nitrocellulose sheets (20). followed by goat anti-human IgG-peroxidase conjugate (Sig- Recombinant T. cruzi Antigen. The recombinant T. cruzi ma) and developed with O-phenylenediamine chromogen. For clone B13 was originally isolated from a Agtll expression epitope mapping of B13 protein, we adapted competitive library, as described (22). The insert of B13 was subcloned in ELISA protocols (13, 14). Briefly, appropriate dilutions ofsera vector pMSgtll, in phase with the lacZ gene. The expressed (yielding OD values in the 0.35-0.6 range) were preincubated fusion protein B13 and ,B-galactosidase were affinity-purified with 1, 10, or 100,uM of each synthetic peptide for 1 hr at 37GC. on p-aminophenyl-f3-D-galactopyranoside-agarose columns. Each serum/peptide mixture was then incubated overnight in B13 recombinant protein contains 16 tandemly repeated 12- triplicate wells of a B13 protein-sensitized polyvinyl chloride amino acid motifs. A rabbit antiserum to B13 protein was microtiter plate (50 ng per well). The reaction was developed obtained as described (22). as described above. The percentage inhibition of serum bind- Synthetic Peptides. Peptides were synthesized with the ing to solid-phase B13 protein by each peptide at 100 ,uM "teabag" method (23), HPLC-purified, and sequenced. Table concentration was normalized to allow inter-test comparisons 1 lists the peptides used, including a 19-mer that encompasses according to the equation: all possible linear epitopes (24) within the 12-mer tandem repeats of B13 protein (pB13) (22). In addition, 9-mer B13 % inhibition = [1 - (OD of peptide peptides (S1-6) were designed in "sliding window" fashion (25) for epitope mapping. Peptides pl-20 and p1439-1453 are - OD of pB13/OD of NR - OD of pB13)] x 100. derived from the sequence of human cardiac myosin HC, (3 isoform, and contain B13-homologous sequences (bold-faced Statistical Methods. Fisher's exact test was used to evaluate type). The nonrelated peptide (NR) represents residues 22-41 frequency differences between different patient groups. Stu- of the human T-cell receptor variable-region a-subunit 13 dent's t test was used to compare means of OD values in ELISA chain. tests between different groups of serum. The MACAW program Affinity Purification of Anti-Cardiac Myosin HC Antibod- (27) was used for protein-sequence alignment. ies (AMA). Immunoselection of specific antibodies was done as described (26). After adequate blocking, myosin HC im- RESULTS munosorbents (50 ,ug of myosin per strip) were incubated overnight at 4°C with 1.5 ml of a 1:10 dilution of each serum Prevalence of Anti-Cardiac Myosin IgG Antibodies in Pa- sample. Bound antibodies were eluted with 0.02 M glycine, pH tients' and Normal Sera. All 45 sera from CCC, ASY, and 2.8, neutralized with 1 M Tris, pH 8.6, and reconstituted to normal groups were reactive when tested in a purified human Tris-buffered saline/1% nonfat milk. Each AMA preparation cardiac ELISA. The distribution of reactivities was compara- was tested for specific anti-myosin HC IgG antibody activity by ble, and mean ODs (horizontal bars) in each group were not incubation with human heart lysate immunoblots. significantly different by Student's t test (Fig. 1). In addition, Immunoblots. Immunoblots were done as described (20). all sera recognized the major 210-kDa band-comigrating Briefly, electroblotted nitrocellulose strips were incubated with myosin HC-in human heart lysate immunoblots (data overnight at 4°C with either AMA obtained from individual not shown). sera or 1:40 dilutions of unfractionated sera, followed by goat 1.8 Table 1. 0 Synthetic peptides 1.6 Peptides 1.4]- B13-derived pB13 GDKPSLFGQAAAGDKPSLF 1.2- Si a 0 GDKPSLFGQ 0 1- S2 KPSLFGQAA 0 S3 SLFGQAAAG 0.8- 0 I1 S4 FGQAAAGDK 0.6- 0 S5 QAAAGDKPS 0.4 0 S6 AAGDKPSLF I Human cardiac myosin 0.2iI i S B HC-derived un aYIV pl-20 MGDSEMAVFGAAAPYLRKSE Normal ASY CCC p1439-1453 NAAAAALDRKQRNFD (n=10) (n=11) (n=24) Human T-cell receptor Va chain FIG. 1. ELISA with purified human cardiac myosin as the solid- NR phase antigen incubated with 1:200 dilution of sera from normal, ASY, NKSAKQFSLHIMDSQPDGS and CCC individuals. A 1:200 dilution was found to yield OD values Va, variable region a. in the most informative range. Horizontal lines represent mean values. Downloaded by guest on September 27, 2021 Immunology: Cunha-Neto et at Proc. Natl. Acad. Sci. USA 92 (1995) 3543 B the pl-20 peptide, competes for binding of the anti-B13 A antibodies in serum of a CCC patient in a dose-dependent fashion. On the other hand, neither myosin peptide competes a b for binding of the antibodies in a ASY serum (Fig. 3B). Apart from the myosin-crossreactive epitope, the B13 pro- kDa .f: tein repeat motif is known to contain an immunodominant T. by unfractionated sera from 97% of 210 cruzi epitope, recognized fl T. cruzi-infected individuals irrespective of disease manifesta- tion (22, 29). To map this immunodominant epitope, synthetic 140 31. 9-mer peptides derived from B13 were also tested. Fig. 5 116 )0- indicates that only the 9-mer S4 peptide, apart from the 19-mer 97 pB13 peptide, completely inhibits the binding of both CCC serum (Fig. 4A) and ASY serum (Fig. 4B) to B13 recombinant 68 protein. The p1439-1453 myosin peptide, along with pB13 and S4 B13-derived peptides, consistently inhibits the binding of CCC anti-B13 antibodies in six CCC sera in B13 ELISA (Fig. 5). Three other ASY sera were tested, corroborating that only B13-derived peptides pB13 and S4 inhibit ASY serum antibody binding to B13 (data not shown). At low dilutions (1:40-1:160), FIG. 2. (A) Immunoblot with T. cruzi trypomastigote lysate incu- sera from 10 normal controls and 10 cardiopathic Duchenne bated with AMA purified from a CCC serum (strip a) and a rabbit in the antiserum to B13 recombinant protein (strip b); molecular mass muscular dystrophy patients displayed OD readings markers (kDa) are shown at left. Arrowheads indicate 140/116-kDa range of 0.2-0.3. Such OD readings in B13 ELISA could not bands. (B) Dot-blots with 50 ng of B13 recombinant protein incubated be inhibited by preincubation with peptides p1439-1453 and with AMA immunoselected from 14 CCC sera (strip a) and 14 ASY pB13, thus demonstrating that Duchenne muscle dystrophy sera (strip b). The positions where protein and antibody samples were patient sera and normal sera are devoid of specific anti-B13 applied are indicated with dashes. antibodies (data not shown). Identification of a Cardiac Myosin HC Crossreactive T. cruzi Antigen. Trypomastigote lysate immunoblots were DISCUSSION probed with AMA individually purified from 23 CCC and 11 In this study, we identify the myosin crossreactive T. cruzi ASY sera. A doublet of bands of 140 and 116 kDa (Fig. 2A, antigen B13, and we have mapped the crossreactive epitope to strip a) was recognized by AMA from 14 of 23 (61%) CCC a cardiac myosin-specific 6-amino acid region. The almost patients, in contrast to 1 of 11 (9%) ASYs. The difference in crossreactive recognition among AMA from CCC and ASY 0.4 sera is statistically significant (P = 5.1 X 10-3, Fisher's exact A test). AMA from normal individuals did not recognize the 0.35 doublet of T. cruzi bands. Furthermore, AMA from chagasic 0.3 _ or normal do not bind to LLC-MK2 cell line patients subjects -- antigens in immunoblots (data not shown). 0.25 -1- "17'N-, Fig. 2A, strip b, shows the same 140/116-kDa doublet g 0.2 34 p10NR recognized by a rabbit antiserum to the recombinant T. cruzi IU.15n + protein B13 (22). We then investigated whether AMA from bpl-20 chagasic patients recognized B13 immobilized in dot blots. Fig. 0.1 jk_ 2B indicates that AMA from 14 of 14 CCC sera (100%) and 0.05 - F.;E- 2 of 14 ASY sera (14%) give a positive reaction. The difference \pl439 - in binding to B13 between AMA from CCC and ASY sera is 0 l l~i 1453 1 J,M lOOulLM highly significant (P = 2.3 x 10-6, Fisher's exact test). Cross- 10,lM reactivity in CCC AMA was not secondary to higher levels of 0.6 _ antimyosin or anti-B13 antibody levels, as they were compa- B rable in both CCC and ASY unfractionated sera groups, as 0.5 assessed by ELISA tests (data not shown). AMA from a NR limited number of ASY individuals with myocarditis at en- nu.-4A domyocardial biopsy also recognized B13 in dot-blots (data not n 'AL ~ pl-20 shown). ,B-Galactosidase, the fusion protein support, was not 0 U.J, ------p1439 - recognized by AMA from any of the sera. 1453 Mapping of the Immunodominant and Cardiac Myosin 0.2 HC-Crossreactive B13 Epitopes. To identify regions of pri- mary sequence homology between B13 and cardiac myosin 0.1 that could harbor a crossreactive epitope, the sequence of the pB13 .. II B13 repetitive motif was aligned (27) to the f3 isoform of 10,lM 100 human cardiac myosin HC, the prevalent isoform in human ,uM ventricle (28). Two hexapeptide regions of 80% homology FIG. 3. Competitive ELISA for B13 protein. Different concentra- were disclosed, at positions 8-13 (VFGAAA vs. B13 tions of synthetic peptides derived from human cardiac myosin HC ,3 FGQAAA) and 1442-1447 (AAALDK vs. B13 AAAGDK), isoform (pl-20 and p1439-1453), B13 protein (pB13), and human span- T-cell receptor variable-region a chain (NR) were preincubated with respectively, of cardiac myosin HC. Synthetic peptides serum from CCC patient WA (A) or ASY CA (B). Each mixture was ning the B13-homologous sequences in cardiac myosin HC added to wells of a microtiter plate sensitized with B13 protein. pB13 (pl-20 and p1439-1453) were incubated with CCC and ASY and NR stand as positive and negative competition controls, respec- sera in competitive ELISA experiments with B13 as the tively. Resulting OD values are displayed for each peptide concentra- solid-phase antigen. Fig. 3A shows that p1439-1453, but not tion. Downloaded by guest on September 27, 2021 3544 Immunology: Cunha-Neto et at Proc. Natl. Acad Sci. USA 92 (1995)

n A- V.-4J K`> 0.41"A oS1 0.35------\o~- -- S2 39E-m-

0.3- --cS3 - Z:Z_ ] w -4.-54 0 0.25- .,. 4i.! - m 0 0.2- -S6 .2 -x- pB13 NR a 0.15- ._2 --- NR ._ 0.1 .,As IAC.-1 _ x pB13 Sl S2 S3 S4 S5 S6 pl-20p1439- Ul- 1 pLM 10 AM 100 JIM 1453 FIG. 5. Epitope mapping of the B13 repeat motif. Competitive inhibition of the binding of anti-B13 antibodies in B13 ELISA by synthetic peptides at 100 ,uM concentration. The percentage of inhibition of serum antibody-binding solid-phase B13 protein was calculated according to the equation in Materials and Methods. Inhi- bition values for each peptide were averaged from six CCC sera after I normalization. Fig. 2B, strip b, could also present subclinical Chagas myo- 0 carditis. The presence of two distinct epitopes in the B13 repeat motifs was suspected in face of the findings of restricted recognition of B13 by AMA from CCC sera (Fig. 2B) and unrestricted recognition of an "immunodominant" epitope by pB13 unfractionated serum in B13 ELISA serodiagnosis tests (22, 29). Epitope mapping with competitive inhibition of B13 1 ,M 10 ,uM 100 ,uM ELISA (Figs. 3-5) suggested the existence of two distinct FIG. 4. Competitive ELISA for B13 protein. Different concentra- specificities of B13-binding antibodies: (i) antibodies inhibited tions of synthetic peptides (S1-S6 and pB13, compare with Table 1) by B13 peptides pB13 or S4, found in unfractionated sera from covering B13 linear epitopes, as well as peptides derived from T-cell all T. cruzi-infected individuals tested and possibly responsible receptor variable-region a chain (NR), were preincubated with serum for the binding of B13 protein in serodiagnosis tests, recog- from CCC patient WA (A) or from ASY CA (B). Assays were done nizing the immunodominant epitope contained in B13 peptide as described in the Fig. 3 legend. S4; and (ii) antibodies inhibited by the cardiac myosin peptide p1439-1453, found mainly in CCC sera and representing the exclusive presence of cardiac myosin HC-B13 140/116-kDa antibodies that recognize B13 protein and the 140/116-kDa crossreactive antibodies in CCC sera (Fig. 2) suggests that doublet in AMA preparations, indicating the sequences cardiac myosin HC-B13 crossreactive recognition may be AAALDK in myosin and AAAGDK in B13 as the respective biologically relevant to the pathogenesis of CCC. crossreactive epitopes. B13 peptide S5 (QAAAGDKPS) was The universal reactivity of sera from CCC, ASY, and normal unable to inhibit antibody binding to B13 in CCC sera (Figs. controls against human cardiac myosin both in immunoblots 3-5), which may indicate that residues neighboring the cross- and in ELISA (Fig. 1) indicates the existence of natural reactive hexapeptide also play a role in antibody reactivity. antimyosin antibodies. Although "natural" anti-cardiac myo- The characterization of the p1439-1453 myosin peptide sin antibodies have already been reported in mice (30) and containing the AAALDK hexapeptide homologous to B13 humans (31), our data establish their finding at high preva- AAAGDK as the crossreactive epitope established a structural lence. Our results show a qualitative difference between basis for myosin-B13 crossreactivity. The AAALDK 1442- "cardiopathy-related" anti-myosin antibodies in CCC sera and 1447 myosin sequence is strictly conserved only among cardiac "natural" anti-myosin antibodies in ASY sera (Fig. 2), match- myosin HC isoforms (Fig. 6). Thus, the B13-crossreactive ing the findings in experimental murine infection (14). The myosin epitope recognized by serum antibodies of CCC pa- presence of crossreactive antibodies in sera from ASYs with tients is cardiac myosin HC-specific. The fact that the cross- subclinical Chagas myocarditis may indicate that such anti- reactive epitope in B13, AAAGDK, is embedded into the bodies are associated to Chagas myocarditis per se rather than immunodominant epitope FGQAAAGDK (S4 peptide) is to extensive heart damage. It is tempting to speculate whether puzzling, as ASY sera recognize the latter and ignore the the two ASYs whose sera displayed crossreactive antibodies in former. This finding suggests that production of cardiac myosin

human cardiac 1428IEDLMVDVERSNAAAAALDDEKQRNFDKILAE1458 human cardiac a human adult skeleletal V .. .. I .. . .T . . .C ...... T ......

rabbit smooth muscle LD. .V. .LDNQRQLVSN.E ... KK. .L...

human nonmuscle LD. .L. .LDHQRQS.CN.E .. .KK. .QL ... FIG. 6. Alignment of the 1428-1458 region of human cardiac myosin HC containing the B13-crossreactive epitope with other tissue-specific myosin HC isoforms. Genenbank peptide locus codes are as follows: human cardiac ,B myosin HC, HUMBMYH7CD_1; human cardiac a myosin HC, HUMCAMHC 1; human adult skeletal myosin HC, HSMYH1R 1; rabbit uterus smooth muscle myosin HC, S68021 1; and human nonmuscle myosin HC, HUMMYONM_1. Downloaded by guest on September 27, 2021 Immunology: Cunha-Neto et at Proc. Natl. Acad Sci. USA 92 (1995) 3545 HC-B13 crossreactive antibodies in CCC may be secondary 4. Andrade, Z. (1958) Rev. Goiana Med. 4, 103-119. to selective recognition of the crossreactive sequences 5. Teixeira, A., Teixeira, M. L. & Santos-Buch, C. (1975) Am. J. AAAGDK/AAALDK, under immune response gene control. Pathol. 80, 163-180. Thus, it can be hypothesized that crossreactive recognition- 6. Cossio, P. M., Diez, C., Szarfman, A., Kreutzer, E., Candiolo, B. & Arana, R. M. (1974) Circulation 49, 13-21. and the attending heart damage of CCC-would only occur in 7. Levin, M. J., Mesri, E., Benarous, R., Levitus, G., Schijman, A., T. cruzi-infected individuals endowed, for instance, with per- Torres, H. & Segura, E. (1989) Am. J. Trop. Med. Hyg. 41, missive HLA alleles. Family clustering (32) and HLA associ- 530-538. ation (33, 34) studies support a possible role for immunoge- 8. Kerner, N., Liegeard, P., Levin, M. J. & Joskowicz, M. H. (1991) netic components in the differential susceptibility of the Exp. Parasitol. 73, 451-459. 20-30% of T. cruzi-infected individuals who develop CCC. The 9. Van Voorhis, W., Schelekewy, L. & Trong, H. (1991) Proc. Natl. absence of crossreactive antibodies in cardiopathic Duchenne Acad. Sci. USA 88, 5993-5997. muscular dystrophy patients' sera (data not shown) suggests 10. Rizzo, L. V., Cunha-Neto, E. & Teixeira, A. (1989) Infect. Immun. 57, 2640-2644. that such antibodies may not be formed as a consequence of 11. Harrington, W. F. & Rodgers, M. E. (1984)Annu. Rev. Biochem. heart damage alone. However, it cannot be formally discarded 53, 35-73. that heart damage in a T. cruzi-infected individual might raise 12. Dale, J. B. & Beachey, E. H. (1986) J. Exp. Med. 164, 1785-1790. such crossreactive antibodies. 13. Neu, N., Beisel, K., Traytsman, M., Rose, N. & Craig, S. (1987) In spite of the 100% association with CCC patients, direct J. Immunol. 138, 2488-2492. participation of the myosin-B13 crossreactive antibody in CCC 14. Tibbetts, R. S., McCormick, T. S., Rowland, E. C., Miller, S. D. pathogenesis is not likely. The bulk of evidence supports the & Engman, D. M. (1994) J. Immunol. 152, 1493-1499. participation of T cells rather than antibody in heart damage 15. Neu, N., Rose, N., Beisel, K., Herskowitz, A., Gurri-Glass, G. & (2, 3, 5, 10, 35). The association of cardiac myosin HC-B13 Craig, S. (1987) J. Immunol. 139, 3630-3636. 16. Smith, S. & Allen, P. (1991) J. Immunol. 147, 2141-2147. crossreactive antibodies with CCC may be only signaling the 17. Higuchi, M. L., Floriano de Morais, C., Pereira Barreto, A. C., presence of "helper" T cells of similar specificity, these ones Lopes, E. A., Stolf, N., Bellotti, G. & Pileggi, F. (1987) Clin. potentially involved in heart tissue damage. The identification Cardiol. 10, 665-669. of a defined heart-specific epitope (AAALDK) of a major 18. Margossian, S. & Lowey, S. (1982) Methods Enzymol. 85, 55-71. heart protein (cardiac myosin HC) that is antigenically mim- 19. Laemmli, U. K. (1970) Nature.(London) 227, 680-685. icked by a secondary epitope (AAAGDK) of an immunodom- 20. Towbin, H., Staehelin, L. & Gordon, J. (1979) Proc. Natl. Acad. inant T. cruzi antigen (B13) is in itself a corollary of the theory Sci. USA 76, 4350-4354. of autoimmune pathogenesis of CCC. The demonstration that 21. Zingales, B., Andrews, N. W., Kuwajima, V. Y. & Colli, W. to (1982) Mol. Biochem. Parasitol. 6, 111-124. crossreactive antibody recognition is mainly restricted CCC 22. Gruber, A. & Zingales, B. (1993) Exp. Parasitol. 76, 1-12. patients defines human cardiac myosin HC and T. cruzi protein 23. Houghten, R. A. (1985)Proc. Natl. Acad. Sci. USA 82, 5131-5135. B13 as an antigen pair of potential relevance to the pathogen- 24. Kabat, E. A. (1970) Ann. N.Y Acad. Sci. 169, 43-54. esis of heart lesions in human CCC. Specific treatment with 25. Geysen, H. M., Rodda, S. J., Mason, T., Tribbick, G. & Schoofs, anti-T cruzi drugs may not abort the progression of CCC P. (1987) J. Immunol. Methods 102, 259-274. among T. cruzi-infected individuals (36). Thus, alternative 26. Tovey, E. & Baldo, B. (1989) in Protein Blotting, eds. Baldo, B., antigen-specific immunosuppression approaches blocking the Tovey, E. & St. Leonards, N. (Karger, Basel), pp. 43-64. deleterious autoimmune response without interfering with 27. Schuler, G. D., Altschul, S. F. & Lipman, D. J. (1991) Proteins antiparasite immunity may help control heart damage in Struct. Funct. Genet. 9, 180-190. 28. Gorza, L., Mercadier, J.-J., Schwartz, K., Thornell, L. E., Sartore, Chagas disease. Further, our findings may allow the search for S. & Schiaffino, S. (1984) Circ. Res. 54, 694-702. effective subunit vaccines devoid of components of T. cruzi 29. Tropical Disease Research/World Health Organization (1990) that bear the pathogenic heart-crossreactive determinants. Mem. Inst. Oswaldo Cruz 85, 487-543. 30. Berneman, A., Ternynck, T. & Avrameas, S. (1992) Eur. J. We thank Drs. A. C. Pereira-Barreto and B. Ianni for allowing Immunol. 22, 625-633. access to patients. We are indebted to Dr. A. C. Goldberg for critically 31. Cunningham, M. W., McCormack, J. M., Talaber, L. R., Harley, reviewing the manuscript. This work has been supported by grants J. B., Ayoub, E. M., Muneer, R. S., Chun, L. T. & Reddy, L. 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