Molecular mimicry in the autoimmune pathogenesis of rheumatic heart disease

Molecular mimicry is a hallmark of the pathogenesis of rheumatic fever where the streptococcal group A carbohydrate epitope, N-acetyl glucosamine, and the a-helical coiled-coil streptococcal M protein structurally mimic cardiac myosin in the human disease, rheumatic carditis, and in animal models immunized with streptococcal M protein and cardiac myosin. Recent studies have unraveled the potential pathogenic mechanisms by which the immune response against the group A streptococcus attacks the rheumatic valve leading to chronic rheumatic heart disease. Both B- and T-cell responses are involved in the process, and evidence for the hypotheses of molecular mimicry and epitope spreading are reviewed.     

T cell mimicry in inflammatory heart disease

Inflammatory heart diseases such as myocarditis and rheumatic heart disease result from the infiltration of the myocardium or valve with T cells and macrophages that result in scarring of the myocardium or valve and alteration in cardiac function. Our studies of T cells from these diseases have identified cardiac myosin in both rheumatic carditis and myocarditis as an important autoantigen. In rheumatic heart disease, streptococcal M protein specific T cells migrate to valves. By investigating streptococcal M protein and cardiac myosin in the Lewis rat model of myocarditis and valvulitis, T cell mimicry is supported as a potential mechanism in disease. Structural and immunological mimicry between the streptococcal M protein and cardiac myosin is shown directly in the Lewis rat model. Rat T cell lines demonstrate mimicry between cardiac myosin and M protein, and T cells isolated directly from inflammatory lesions in myocarditis respond to streptococcal M protein peptides. Studies in BALB/c mice also support the immunological crossreactivity of T cells primed against cardiac myosin with streptococcal M protein peptides containing cardiac myosin homologies. T cell lines produced from the Lewis rat specific to the cardiac myosin like sequences of streptococcal M protein migrated to the valves after passive transfer of the M protein specific T cell lines. In coxsackieviral myocarditis in the MRL mouse strain, cardiac myosin mimicking M protein peptide NT4 was found to induce tolerance and prevent coxsackieviral induced myocarditis, suggesting T cell mimicry between coxsackievirus and streptococcal M protein, both of which are associated with inflammatory heart disease. T cell mimicry between cardiac myosin and microbial antigens such as the streptococcal M protein may prime the immune system for inflammatory heart disease.     

Induction of Myocarditis and Valvulitis in Lewis Rats by Different Epitopes of Cardiac Myosin and Its Implications in Rheumatic Carditis

Immune responses against cardiac myosin and group A streptococcal M protein have been implicated in the pathogenesis of rheumatic heart disease. Although cardiac myosin is known to produce myocarditis in susceptible animals, it has never been investigated for its role in production of valvular heart disease, the most serious sequelae of group A streptococcal infection in acute rheumatic fever. In our study, cardiac myosin induced valvulitis in the Lewis rat, and epitopes responsible for production of valvulitis were located in the rod region. Human and rat cardiac myosins induced severe myocarditis in the Lewis rats as expected. A purified S2 fragment (amino acid sequences 842 to 1295) produced the most severe myocarditis as well as valvulitis. Different regions of light meromyosin produced valvulitis (residues 1685 to 1936) or myocarditis (residues 1529 to 1611). Because streptococcal M proteins produced valvular heart disease in Lewis rats and have been linked to anti-cardiac myosin responses, we reacted myosin-sensitized lymphocytes isolated from the hearts of Lewis rats with peptides of streptococcal M5 protein in tritiated thymidine assays. Infiltrating lymphocytes responded most strongly to peptides within the B repeat region of streptococcal M protein. These data show direct evidence that immune responses against cardiac myosin lead to valvular heart disease and the infiltration of the heart by streptococcal M protein reactive T lymphocytes.     

Induction of autoimmune valvular heart disease by recombinant streptococcal m protein

Rheumatic heart disease is an autoimmune sequela of group A streptococcal infection. Previous studies have established that streptococcal M protein is structurally and immunologically similar to cardiac myosin, a well-known mediator of inflammatory heart disease. In this study, we investigated the hypothesis that streptococcal M protein could produce inflammatory valvular heart lesions similar to those seen in rheumatic fever (RF). Fifty percent (3 of 6) of Lewis rats immunized with recombinant type 6 streptococcal M protein (rM6) developed valvulitis as well as focal lesions of myocarditis. Valvular lesions initiated at the valve surface endothelium spread into the valve. Anitschkow cells and verruca-like lesions were present. T cells from rM6-immunized rats proliferated in the presence of purified cardiac myosin, but not skeletal myosin. A T-cell line produced from rM6-treated rats proliferated in the presence of cardiac myosin and rM6 protein. The study demonstrates that the Lewis rat is a model of valvular heart disease and that streptococcal M protein can induce an autoimmune cell-mediated immune attack on the heart valve in an animal model. The data support the hypothesis that a bacterial antigen can break immune tolerance in vivo, an important concept in autoimmunity.     

Molecular analysis of human cardiac myosin-cross-reactive B- and T-cell epitopes of the group A streptococcal M5 protein.

The group A streptococcal M protein is an important virulence determinant eliciting protective and autoimmune responses against the streptococcus and cardiac myosin, respectively. In this report, the major human cardiac myosin-cross-reactive T-cell epitopes of M5 protein are identified and localized to myosin-like repeats within the M5 molecule. BALB/c mice were immunized with human cardiac myosin, and the dominant myosin-cross-reactive T-cell epitopes of M5 protein were identified with a panel of 23 overlapping peptides spanning the A, B, and C repeat regions of M5 protein. Human cardiac myosin-cross-reactive T-cell epitopes of M5 protein were localized to several sequences in the M5 peptides NT4 (GLKTENEGLKTENEGLKTE), NT5 (KKEHEAENDKLKQQRDTL), B1B2 (VKDKIAKEQENKETIGTL), B2 (TIGTLKKILDETVKDKIA), B3A (IGTLKKILDETVKDKLAK), and C3 (KGLRRDLDASREAKKQ). The NT4 repeated sequence LKTEN was highly homologous with a site conserved in cardiac myosins, the B repeat region peptides were 47% homologous to human cardiac myosin amino acid sequence, and the C3 sequence RRDL was identical to a highly conserved site in skeletal and cardiac myosins. Immunization of BALB/c mice with each of the overlapping M5 peptides revealed myosin-cross-reactive B-cell epitopes throughout the A and C repeat regions and one major epitope in the B repeat region containing the previously reported Gln-Lys-Ser-Lys-Gln (QKSKQ) epitope. The data suggest that the M5 peptides elicited higher antibody titers to cardiac myosin than to skeletal myosin and that several sites in the A and B repeat regions of M5 protein induced myocardial inflammatory infiltrates.     

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.     

How an autoimmune reaction triggered by molecular mimicry between streptococcal M protein and cardiac tissue proteins leads to heart lesions in rheumatic heart disease

Molecular mimicry between microbial antigens and host tissue is suggested as a mechanism for post-infectious autoimmune disease. In the present work we describe the autoimmune reactions of two severe rheumatic heart disease (RHD) patients, through an analysis of heart-infiltrating T-cell repertoire, antigen recognition, and cytokine production induced by specific antigens. T-cell clones derived from oligoclonally expanded T cells in the heart cross-recognized M5 peptides, heart tissue-derived proteins, and myosin peptides. We show, using binding affinity assays, that an immunodominant streptococcal peptide (M5(81-96)) is capable of binding to the HLA-DR53 molecule. The same peptide was recognized by an infiltrating T-cell clone from a patient carrying HLA-DR15, DR7, and DR53 molecules. This suggests that this peptide is probably presented to T cells in the context of the HLA-DR53 molecule. Cross-reactive heart-infiltrating T cells activated by the M5 protein and its peptides and by heart tissue-derived proteins produced predominantly inflammatory cytokines. Interleukin (IL)-4 was produced in small amounts by mitral valve intralesional T-cell lines and clones. Altogether, these results suggest that mimicry between streptococcal antigens and heart-tissue proteins, combined with high inflammatory cytokine and low IL-4 production, leads to the development of autoimmune reactions and cardiac tissue damage in RHD patients.     

Evidence for actinlike proteins in an M protein-negative strain of Streptococcus pyogenes.

Antigens shared between Streptococcus pyogenes and heart tissue may play an important role in autoimmune cardiac injury associated with acute rheumatic fever. Antiheart/antistreptococcal antibodies found in the disease react with antigens of S. pyogenes, including M protein and a 60-kDa antigen distinct from M protein. Heart antigens recognized by these cross-reactive antistreptococcal antibodies include myosin and actin. To investigate the presence of a streptococcal actin, established protocols for the polymerization and isolation of eukaryotic actin were used to extract and concentrate actinlike proteins from M- streptococcal cells. The polymerized bacterial actin from the streptococcal extract was probed in immunoblots with an antiactin monoclonal antibody. Two proteins of about 60 kDa in the polymerized bacterial actin reacted with the antiactin antibody. Proteins in the polymerized bacterial actin extract of about 43 and 60 kDa behaved like eukaryotic actin by binding to myosin and DNase I affinity columns. Filaments were demonstrated by electron microscopy in the polymerized bacterial actinlike extract, which also enhanced the ATPase activity of eukaryotic myosin. The data suggest that proteins resembling actin are present in S. pyogenes.     

B- and T-Cell Responses in Group A Streptococcus M-Protein- or Peptide-Induced Experimental Carditis

The etiology of rheumatic fever and rheumatic heart disease (RF/RHD) is believed to be autoimmune, involving immune responses initiated between streptococcal and host tissue proteins through a molecular mimicry mechanism(s). We sought to investigate the humoral and cellular responses elicited in a Lewis rat model of group A streptococcus M-protein- or peptide-induced experimental valvulitis/carditis, a recently developed animal model which may, in part, represent human rheumatic carditis. Recombinant streptococcal M5 protein elicited opsonic antibodies in Lewis rats, and anti-M5 antisera recognized epitopes within the B- and C-repeat regions of M5. One peptide from the streptococcal M5 protein B-repeat region (M5-B.6, amino acids 161 to 180) induced lymphocytes that responded to both recombinant M5 and cardiac myosin. Rats immunized with streptococcal M5 protein developed valvular lesions, distinguished by infiltration of CD3⁺, CD4⁺, and CD68⁺ cells into valve tissue, consistent with human studies that suggest that RF/RHD are mediated by inflammatory CD4⁺ T cells and CD68⁺ macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD.There is a wealth of evidence to indicate that the immunopathogenic mechanisms in rheumatic heart disease involve autoimmunity as a result of molecular mimicry between streptococcal and host tissue proteins (7, 16), although the precise mechanisms are not completely understood. Progress has been made in this area of research by analyzing cellular and humoral responses of peripheral blood samples from rheumatic fever and rheumatic heart disease (RF/RHD) patients (11, 15, 17, 19). However, study of T cells and associated cytokines involved in initiating tissue damage is required. Limited access to tissue samples from RF/RHD patients is a major obstacle to these studies, and an acceptable animal model would facilitate further studies. An animal model, in which the immunopathological mechanisms or outcome of disease resembles those that occur in humans, is a logical adjunct to human studies.For many years, attempts to establish a suitable animal model for RF/RHD had limited success, with none of the proposed models displaying the same pathological changes as those seen in human patients (23). The rat autoimmune valvulitis (RAV) model, developed by Quinn and colleagues (30) whereby Lewis rats immunized with recombinant streptococcal M protein develop hallmark RHD lesions in heart valves, has shown promise as a suitable animal model of rheumatic carditis.A role for molecular mimicry in RF/RHD immunopathogenesis has also been supported by the study of Quinn (30) and by others using the RAV model (14). Peripheral blood T-cell lines from M-protein-immunized rats proliferated in response to cardiac myosin (30), and T cells from heart lesions of cardiac myosin-immunized rats also responded to peptides from the B-repeat region of M protein (14). The RAV model has also been used in our laboratory to induce valvulitis/carditis by immunizing Lewis rats with C-terminal M-protein peptides (26).In this study, B- and T-cell responses in Lewis rats immunized with group A streptococcus (GAS) M5 protein or selected M5 peptides were examined to further validate the use of the RAV model as a suitable animal model for RF/RHD. Immunostaining of cellular infiltrates in valvular and myocardial tissue revealed that heart damage observed in streptococcal M-protein-immunized rats is mediated by CD4⁺ T cells and macrophages, in agreement with human studies (19).     

Reactivity of rheumatic fever and scarlet fever patients' sera with group A streptococcal M protein, cardiac myosin, and cardiac tropomyosin: a retrospective study

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.     

Rheumatic Fever,Autoimmunity, and Molecular Mimicry: The Streptococcal Connection

The group A streptococcus, Streptococcus pyogenes, and its link to autoimmune sequelae, has acquired a new level of understanding. Studies support the hypothesis that molecular mimicry between the group A streptococcus and heart or brain are important in directing immune responses in rheumatic fever. Rheumatic carditis, Sydenham chorea and a new group of behavioral disorders called pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections are reviewed with consideration of autoantibody and T cell responses and the role of molecular mimicry between the heart, brain and group A streptococcus as well as how immune responses contribute to pathogenic mechanisms in disease. In rheumatic carditis, studies have investigated human monoclonal autoantibodies and T cell clones for their crossreactivity and their mechanisms leading to valve damage in rheumatic heart disease. Although studies of human and animal sera from group A streptococcal diseases or immunization models have been crucial in providing clues to molecular mimicry and its role in the pathogenesis of rheumatic fever, study of human monoclonal autoantibodies have provided important insights into how antibodies against the valve may activate the valve endothelium and lead to T cell infiltration. Passive transfer of anti-streptococcal T cell lines in a rat model of rheumatic carditis illustrates effects of CD4+ T cells on the valve. Although Sydenham chorea has been known as the neurological manifestation of rheumatic fever for decades, the combination of autoimmunity and behavior is a relatively new concept linking brain, behavior and neuropsychiatric disorders with streptococcal infections. In Sydenham chorea, human mAbs and their expression in transgenic mice have linked autoimmunity to central dopamine pathways as well as dopamine receptors and dopaminergic neurons in basal ganglia. Taken together, the studies reviewed provide a basis for understanding streptococcal sequelae and how immune responses against group A streptococci influence autoimmunity and inflammatory responses in the heart and brain.     

Streptococcal mimicry and antibody-mediated cell signaling in the pathogenesis of Sydenham's chorea

Recent evidence suggests that the pathogenesis of Sydenham's chorea following group A streptococcal infection is due to antibodies which develop due to the infection and infiltrate the brain and basal ganglia. Antibodies present in acute chorea react with the surface of neuronal cells and signal the induction of calcium calmodulin dependent protein kinase II with elevation of tyrosine hydroxylase and subsequent dopamine release which may lead to the movement disorder. The antibodies present in disease recognize lysoganglioside and the group A streptococcal epitope, N-acetyl-glucosamine. Monoclonal antibodies (mAbs) from Sydenham's chorea demonstrated the mimicry between lysoganglioside and the group A streptococcal carbohydrate epitope. A group of antibodies present in pediatric autoimmune neuropsychiatric disorders (PANDAS) were similar but not identical to the antibodies observed in chorea.     

Comprehensive analysis of antibody responses to streptococcal and tissue antigens in patients with acute rheumatic fever

Acute rheumatic fever (ARF) is an autoimmune disease occurringin individuals following untreated group A streptococcal infectionbelieved to be triggered by antibodies to bacterial componentsthat cross-react with human tissues. We developed a multiplexedimmunoassay for the simultaneous quantitation of antibodiesto nine streptococcal-related antigens including streptolysinO (SLO), DNase B, collagen I and IV, fibronectin, myosin, groupA carbohydrate, M6 protein and streptococcal C5a peptidase.Utilizing this method, we examined serum from 49 ARF, 58 pharyngitispatients and age- and sex-matched controls in samples collectedat initial disease onset, and at 4 weeks, 6 months and 1 yearafter diagnosis. Antibody responses were significantly higherfor SLO, DNase B, M6 protein, group A carbohydrate and the cross-reactiveantigens collagen I and myosin in ARF compared with pharyngitispatients (P 0.05). Moreover, we found significantly elevatedantibody responses in the ARF patients with rheumatic heartdisease to fibronectin and collagen I compared with ARF patientswithout heart disease. The major differences between the ARFpatients with and without carditis appear to be in the immuneresponse to the putative heart valve components, collagen Iand fibronectin.     

Rheumatic fever: the T cell response leading to autoimmune aggression in the heart

Molecular mimicry between streptococcal and heart components has been proposed as the triggering factor leading to autoimmunity in rheumatic heart disease (RHD). CD4+ T cells apparently are the ultimate effectors of chronic heart lesions. In this review we summarize the studies on the T cell response of peripheral blood mononuclear cells and T cell clones infiltrating heart lesions from RHD patients against streptococcal M protein peptides and human heart tissue proteins. The T cell receptor usage and the cytokine profile of intralesional mononuclear cells are also presented.     

Immunological Relationship between the Class I Epitope of Streptococcal M Protein and Myosin

The class I epitope of streptococcal M protein is an epidemiological marker for acute rheumatic fever (ARF)-associated serotypes of group A streptococci and is recognized by anti-M protein monoclonal antibody (MAb) 10B6. Using MAb 10B6, we determined the relationship between the class I epitope of M protein and the α-helical coiled-coil protein myosin. MAb 10B6 reacted by enzyme-linked immunosorbent assay and Western blotting with human cardiac myosin and rabbit skeletal myosin and its heavy meromyosin (HMM) subfragment. Overlapping synthetic peptides of M5 protein were used to identify the region of M5 protein recognized by MAb 10B6. Two C repeat peptides (C2A and C3) containing the amino acid sequence KGLRRDLDASREAK reacted with MAb 10B6. Partial sequence identity, RRDL, was found in the HMM fragment of myosin, which reacted with MAb 10B6. However, not all peptides of M5 protein and myosin containing the RRDL sequence reacted with MAb 10B6. ARF sera and sera from uncomplicated pharyngitis (UNC) reacted with C repeat region peptides of M protein, while acute glomerulonephritis sera were not as reactive. Affinity-purified human antibody to peptide C3 reacted with myosin. The data demonstrate that the class I epitope of M protein is immunologically cross-reactive with myosin and the HMM subfragment, and antibodies to peptide C3 and myosin were present in ARF and UNC sera.Acute rheumatic fever (ARF) is an inflammatory disease that can follow group A streptococcal pharyngitis. The most serious clinical manifestation is rheumatic carditis; however, arthritis, chorea, erythema marginatum, or subcutaneous nodules may be present (40, 41). The pathogenesis of ARF is thought to be mediated by autoimmune mechanisms activated during a streptococcal infection (40). The autoimmune hypothesis is supported by a number of previous observations, including a time interval of at least 3 weeks between the initial streptococcal throat infection and the onset of ARF (40, 41), the identification of heart-reactive immunoglobulin (Ig) and complement deposits in the myocardium of patients with fatal rheumatic carditis (25–27, 30), and the elevation of heart-reactive antibodies in the sera of patients with ARF (46). Cardiac myosin has been identified as one of the cardiac antigens recognized by these heart-reactive antistreptococcal autoantibodies (13, 29).Streptococcal M protein, an α-helical coiled-coil protein, structurally and immunologically mimics host tissue antigens, particularly the rod region of myosin (12, 14, 15, 17, 34, 35). Sequence analysis has revealed that streptococcal M proteins contain blocks of internally repeated amino acid sequences referred to as A, B, and C repeat regions (19). The NH2-terminal nonrepeat and A repeat regions contain determinants of type specificity, while epitopes found in the B and more highly conserved C repeat regions may be common to different M serotypes (19). While there are nearly 100 different serological types of group A streptococcal M protein, epidemiological studies indicate that only a limited number of M protein serotypes are associated with ARF outbreaks (6). This finding suggests that certain M protein serotypes may be more rheumatogenic than others. In a previous attempt to classify streptococcal serotypes according to their rheumatogenic capacity, Widdowson identified human antisera directed to a non-type-specific protein moiety of M protein known as M-associated protein (44, 45). However, a more recent classification scheme has been proposed by Bessen and colleagues, in which streptococcal serotypes were grouped based on the expression of a conserved surface-exposed M protein epitope (4). It was demonstrated that the M serotypes associated with the majority of ARF outbreaks possessed an epitope (class I) defined by monoclonal antibody (MAb) probes 10B6 and 10F5. The sequence of the 10B6 and 10F5 epitope was localized to a 15-amino-acid fragment within the C repeat region of the type 6 M protein (23). The remaining serotypes (class II) lack this epitope or the determinant is structurally inaccessible in those strains. There was a close parallel between serotypes designated class I and those serotypes previously classified as M-associated protein I by Widdowson (44, 45). The fact that only certain serotypes within class I streptococci are rheumatogenic implies that these organisms are of a phenotype that is capable of inducing ARF (4). This implication is supported in part by a recent publication in which it was shown that sera of ARF patients contained high levels of antibodies to the class I epitope, suggesting that their disease was the result of an infection by a class I streptococcus (5).Elevated titers of antibodies to many streptococcal antigens (2), including M protein and the self-antigen myosin (12–15, 17, 29), are associated with ARF. While antibodies to M protein are crucial for the opsonization of streptococci, they have also been implicated in the immunological cross-reactions between streptococci and host tissue antigens such as cardiac myosin (12–15, 17, 29). In earlier studies, many of these cross-reactive epitopes have been localized to the N-terminal, hypervariable A and B repeat regions of the M molecule (12, 15, 17). Myosin-reactive antibodies, found to be elevated in almost all cases of ARF (13), have been shown to bind to human heart tissue and to cross-react with streptococcal M protein (12). Previous studies have demonstrated that immunization of animals with the cell walls of certain strains of group A streptococci resulted in the production of heart-reactive antibodies which could be adsorbed with streptococcal extracts containing streptococcal M protein (16, 24, 28). Human MAbs or myosin affinity-purified antibodies produced from patients with ARF cross-reacted with streptococcal M protein and human cardiac myosin and contributed to the presence of heart-cross-reactive antistreptococcal antibodies in ARF (12, 13, 39). More recent studies have identified cytotoxic antistreptococcal/antimyosin MAbs from rheumatic carditis patients (1). Antimyosin antibody has been shown to deposit in the heart tissues of susceptible mice (31), and a cytotoxic mouse antistreptococcal/antimyosin antibody which binds to the surface of heart cells and to the α-helical coiled coil molecule laminin has been described (10).Identification of myosin cross-reactive epitopes of M protein recognized in ARF has been reported for the amino-terminal half of the molecule (12, 15, 17), and a study by Vashishtha and Fischetti demonstrated antimyosin antibody responses to the C repeat region. However, the reactivity was directed only to denatured myosin (43). More recently, studies of the C repeat or carboxy-terminal region of M protein have shown T-cell cross-reactions with myosin (38). The goal of the present study was to investigate the possibility that the class I epitope in the C repeat region of M protein cross-reacts immunologically with myosin. In this study we show that MAb 10B6, which recognizes the class I epitope of M protein, reacts with cardiac and skeletal myosin. This study also demonstrates that ARF and UNC sera react with a site in the conserved C repeat region of M protein within the class I epitope of rheumatogenic M protein serotypes. The new data show that in addition to previously described N-terminal epitopes, the class I epitope of streptococcal M protein is immunologically cross-reactive with myosin.(Portions of this work were presented at the XIII International Lancefield Society Meeting on Streptococci and Streptococcal Diseases at the Pasteur Institute in Paris, France, in September 1996.)     

Pathogenesis of group A streptococcal infections

Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks. emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features. At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesins have been reported, and surface plasmin-binding proteins have been defined. The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation.     

T-Cell-dependent antibody response to the dominant epitope of streptococcal polysaccharide, N-acetyl-glucosamine, is cross-reactive with cardiac myosin

Autoantibodies against myosin are associated with myocarditis and rheumatic heart disease. In this study, the antigenic cross-reactivity of myosin and N-acetyl-glucosamine (GlcNAc), the dominant epitope of Group A streptococcal polysaccharide, was examined. Six antimyosin monoclonal antibodies (MAbs) derived from mice with cardiac myosin-induced myocarditis were characterized. All MAbs cross-reacted with GlcNAc, mimicking a subset of MAbs derived from rheumatic carditis patients that bind both myosin and streptococcal polysaccharide. Variable (V) region gene usage was diverse, with five of six MAb heavy-chain V regions encoded by distinct members of the J558 family and the sixth encoded by a member of the VGAM3.8 family. Light-chain V-region segments were derived from the Vk1, Vk4/5, Vk10, and Vk21 families. These antimyosin, anti-GlcNac MAbs demonstrated several T-cell-dependent features: they were predominantly immunoglobulin G, were encoded by V-region genes expressed late in development, and displayed somatic mutation. A direct correlation between the extent of somatic mutation and the affinity for myosin was observed. Affinity for GlcNAc also increased with the frequency of mutation, demonstrating that affinity maturation can occur simultaneously for both self antigen and foreign antigen. Based on these observations, we immunized mice with GlcNAc coupled to bovine serum albumin and demonstrated that a T-cell-dependent response to GlcNAc leads to antimyosin reactivity. We speculate that the pathogenic antibody response in rheumatic carditis may reflect the conversion of a T-cell-independent response to GlcNAc to a T-cell-dependent cross-reactive response to GlcNAc and myosin.     

Group A streptococcal M-protein specific antibodies and T-cells drive the pathology observed in the rat autoimmune valvulitis model

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune mediated diseases triggered by group A streptococcal (GAS) infections. Molecular mimicry between GAS M-proteins and host tissue proteins has been proposed as the mechanism that initiates autoreactive immune responses in ARF/RHD. However, the individual role of antibodies and T-cells specific for GAS M-proteins in the pathogenesis of autoimmune carditis remains under-explored. The current study investigated the role of antibodies and T-cells in the development of carditis in the Lewis rat autoimmune valvultis (RAV) model by transferring serum and/or splenic T-cells from rats previously injected with GAS recombinant M5 protein. Here we report that serum antibodies alone and serum plus in vitro expanded rM5-specific T-cells from hyperimmune rats were capable of transferring carditis to naïve syngeneic animals. Moreover, the rats that received combined serum and T-cells developed more severe carditis. Recipient rats developed mitral valvulitis and myocarditis and showed prolongation of P-R intervals in electrocardiography. GAS M5 protein-specific IgG reactivity and T-cell recall response were also demonstrated in recipient rats indicating long-term persistence of antibodies and T-cells following transfer. The results suggest that both anti-GAS M5 antibodies and T-cells have differential propensity to induce autoimmune mediated carditis in syngeneic rats following transfer. The results highlight that antibodies and effector T-cells generated by GAS M protein injection can also independently home into cardiac tissue to cross-react with tissue proteins causing autoimmune mediated immunopathology.     

Mapping the minimal murine T cell and B cell epitopes within a peptide vaccine candidate from the conserved region of the M protein of group A streptococcus

The highly conserved C-terminus of the M protein of group A streptococcus (GAS) is a promising vaccine candidate. An epitope within the conserved C-terminus of the M protein, peptide 145 (a 20-mer with the sequence: LRRDLDASREAKKQVEKALE), has been defined which is the target of opsonic antibodies in both humans and mice, and is recognized by the sera of most adults living in areas of high streptococcal exposure. However, due to potential cross-reactivity between T cells stimulated by this region of the M protein and host cardiac myosin, it is critical to define precisely the minimal protective epitopes within p145. Studies have shown that the immunodominant epitope expressed by p145 is conformational, occurring as an alpha-helical coiled-coil. To enable us to map the murine minimal B cell and T cell epitopes within p145, we have used a novel strategy that allowed us to present shorter sequences of p145 in a native-like conformation. The minimal B cell epitope was found to be contained within residues 7-20 of the p145 sequence, and we have shown that mice immunized with this region are able to generate antibodies that bind to and also opsonize the organism GAS. The T cell epitope is located at the N-terminal region of the p145 sequence, residues 3-14. We have managed, therefore, to define a vaccine candidate--a minimal opsonic B cell epitope within the p145 sequence--that does not incorporate a potentially deleterious T cell epitope.      

Significance of PBMC response of rheumatic fever patients and control individuals to immunodominant streptococcal M5 protein epitopes

β-hemolytic streptococcal infection in developing countries still causes thousands of cases of Rheumatic Fever (RF). Molecular mimicry between streptococcal M protein (strep M) and heart components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility, which is linked to different HLA-DR alleles in different populations. In our hands, RF was significantly associated to HLA-DR7/53. Previous work in our lab has shown that heart-infiltrating T cells that simultaneously recognize strep M and heart proteins. Further, such T cells predominantly recognized the 81-103 strep M5 epitope. In this work, we analysed the proliferative response of peripheral blood mononuclear cells of 99 RF patients and 40 normal controls. Eighty-nine of the RF patients were HLA-typed. As among heart-infiltrating T cells, the 81-103 strep M5 protein epitope is the most frequently recognized epitope among RF PBMC (35.4%), against a 7.5% frequency of proliferation among normal controls (p=0.0018, chi square). However, the 81-103 epitope was as frequently recognized by HLA-DR7,53 positive as by negative individuals (45.2% vs 54.8%, respectively). Taken together, the results suggest that the 81-103 strep M5 epitope may be the immunodominant epitope, “promiscuously” recognized by T cells in a genetically diverse population. The demonstration that molecular mimicry is targeted to a discrete immunodominant “promiscuous” epitope in strep M5 may allow the development of a safe anti-streptococcal synthetic vaccine devoid of such epitopes.