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Dissection of Crossreactive Streptococcal M
and Heart Proteins Opens New Ways of
Immunotherapy and Vaccine Design

L. Guilherme*, MsC, PhD; Jorge Kalil**, MD, PhD;
E. Cunha-Neto, MD, PhD; S. E. Oshiro, MsC;
D. Diefenbach da Silva, MsC; K. Faé ,MsC;
A.C. Goldberg, PhD; A.C. Tanaka, MD;
P. Pomerantzeff, MD; M. H. Kiss, MD

* Head of Culture Section of Immunology Laboratory and ** Clinical Immunology and Allergy,
Department of Clinical Medicine, Heart Institute (InCor) InCor, University of São Paulo,
School of Medicine, São Paulo, Brazil

   Molecular mimicry between beta hemolytic group A streptococcal antigens and human proteins mainly heart tissue proteins is postulated as a mechanism leading autoimmunity in RF/RHD patients (1). The M protein is the major component of streptococcal cell surface and more then 80 different serotypes were described (2). The identification of M protein crossreactive epitopes recognized by antibodies and T lymphocytes is very important for construction of a safe anti-streptococcal vaccine. In order to identify the cellular crossreactive response against M protein and heart tissue derived proteins resolved by 1D electrophoresis we first studied T cells infiltrating heart lesions from Rheumatic Heart Disease (RHD) patients. In this study we demonstrated that 7.5% of intralesional T cell clones were able to recognize simultaneously both streptococcal M5 peptides and human heart tissue proteins.

   These crossreactive clones recognized three different regions 1-25, 81-103 and 163-177 of the M5 molecule and myocardium derived proteins with MW > 150 kDa; aortic valve proteins 90-150 kDa and 30-65 kDa (Figure 1) (3).

   Interestingly, T cell reactivity in the periphery of severe RHD patients showed preferential recognition of M5 (81-96) peptide (Pep 6) (46% of patients vs. 8.6% of controls, P=0.0005) and the same heart tissue fractions recognized by heart-infiltrating T cell clones (4). The nature of heart proteins involved in the crossreactions is currently under investigation using bidimensional T cell Western method. So far, we have studied the reactivity of 41intralesional T cell clones derived from four severe RHD patients against these proteins. Eleven out of 41 (26.8%) were able to recognize mitral valve proteins in the range of 53-68 kDa and pI 5.12 - 8.24 (Figure 2). The humoral reactivity of pools of sera from a severe RHD patient defined by 2- D Western blotting showed a high diversity of myocardium and mitral valve derived proteins recognized (Diefenbach da Silva , Guilherme et al , in preparation). All these data represent the complexity of the immune responses of RHD patients and certainly will be helpful to dissect the M protein epitopes capable to induce a protective immune response without presenting deleterious crossreactions with human tissues.

   On the other hand, we studied the humoral and cellular reactivities (Figures 3, 4 and 5) of RHD patients and adult normal individuals against overlapping M5 protein peptides (N and C terminal portions). Interestingly, the N-terminal region were preferentially recognized by RHD patients while only two overlapping peptides (Pep4 and Pep 5) were preferentially recognized by normal individuals (Figure 3). C-terminal portion was recognized by sera from RHD patients as well as normal individuals (Figure 4). In contrast, cellular responses of PBMC from severe RHD patients were seen only high dose of antigens were used (Oshiro, Guilherme et al, in preparation). Our hypothesis is that severe RHD patients first trigger a high cross reactive response against N-terminal portion and they are not able to trigger a strong immune response against the C terminal region; consequently, they do not acquire protection. So, we think that by selecting T and B M protein epitopes able to elicit a B and T protective immunogenic responses -while excluding crossreactive epitopes - it will be possible to design a safe and effective vaccine against beta hemolytic group A streptococci.

   Our approach on molecular mimicry between streptococcal M protein epitopes and heart antigens opens the possibility of new ways of immunotherapy for severe RHD patients in the future. The immunodominant M5(81-96) peptide (Pep6, Figure 5) preferentially recognized by severe RHD patients, which displays crossreaction with heart tissue proteins, was able to induce the preferential production of IFN  and TNF  over IL-10 and IL-4 among heart-derived T cell lines from 9 severe RHD patients (Figure 6), in line with the predominant detection of inflammatory cytokines on immunochemistry data from RHD heart tissue (5) (and Guilherme et al, 2001 submitted). These results showed that T1-type cytokine profile was predominantly found in the heart tissue of RHD patients and in vitro assay. So, may be it will be possible to down-modulate the T1 cytokine production using specific drugs or immunological intervention.

   The definition of the crossreactive target heart proteins recognized by a specific T cell population, defined by the TCR alpha and beta chains, can be another way to treat severe RHD patients by T cell vaccination. We showed the presence of several oligoclonal T cell expansions in myocardium and mitral valve-derived T cell lines form severe RHD patients, suggesting that the immune response in the local of the lesions is antigen-driven (5, 6). The preferential use of BVJB families on antigen-specific T cell populations is under investigation by our group.

   We believe that the identification of protective and pathogenic antigens and epitopes, that has been taking place in our lab as well as elsewhere (7,8,9) will lead to the stablishment of new ways to treat severe RHD patients and prevent group A streptococcal infections.


1. Kaplan, M.H., J.D. Frengley. 1969. Autoimmunity to the heart in cardiac disease. Current concepts of the relation of autoimmunity to rheumatic fever, postcardiotomy and postinfarction syndromes and cardiomyoopathies. Am. J. Cardiol.24: 459-473

2. Fischetti, V. 1991. Streptococcal M protein. Sci. Am. : 264(6): 32-39

3. Guilherme L, Cunha-Neto E, Coelho V, Snitcowsky R, Pomerantzeff P.MA, Assis RV, Pedra F, Neumann J, Goldberg A, Patarroyo ME, Pillegi F, Kalil J. 1995. Human heart-infiltrating T-cell clones from rheumatic heart disease patients recognize both streptococcal and cardiac proteins. Circulation. 92(3): 415-420.

4. Guilherme L, Oshiro S.E, Faé K C, Cunha-Neto, E, Renesto G, Goldberg AC, Tanaka A C., Pomerantzeff P.M.A, Kiss M.K, Silva C, Guzman F, Patarroyo M.E, Southwood S, Sette A, Kalil, J. 2001. T cell reactivity against streptococcal antigens in the periphery mirrors reactivity of heart infiltrating T lymphocytes in rheumatic heart disease patients. Infect Immunity, volume 69 (9):5345-5351.

5. Guilherme, L, Cunha-Neto, E., Dulphy, N., Tanaka, AC, Toubert, A., Kalil, J. 2001. Heart - directed autoimmunity: the case of Rheumatic Fever. J Autoimmunity 16: 363-367.

6. Guilherme, L, Dulphy, N., Douay, C, Coelho V., Cunha-Neto, E., Oshiro, S.E., Assis, R.V., Tanaka, A. C., Pomerantzeff, P. M. A., Charron, D., Toubert, A., Kaili, J. 2000. Molecular evidence for antigen-driven immune responses in cardiac lesions of rheumatic heart disease patients. Int Immunol, 12(7), 1064-1073.

7. Dale, JB. 1999. Multivalent group A streptococcal vaccine designed to optimize the immunogenecity of six tandem M protein fragments. Vaccine , 17:193-200.

8. Medaglini D, Pozzi G, King TP, Fischetti VA 1995. Mucosal and systemic immune response to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc. Natl Acad. Sci. USA: 92: 6868-6872.

9. Brandt ER, Teh T, Relf Wa, Hobb RI, Good MF: Protective and nonprotective epitopes from amino termini of M proteins from Australian aboriginal isolates and reference strains of group A streptococci. Infect Immunity 2000; 68(12): 6587-94.



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2nd Virtual Congress of Cardiology

Dr. Florencio Garófalo
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