Molecular immunological approach to rheumatic fever vaccine development

Abstract

Group A streptococci cause many diseases and sequelaes worldwide. Their M protein play important roles in these sequelaes. The aim of this thesis is to identify T and B cell protective and cross-reactive epitopes on conserved region of the M protein of Group A s-haemolytic streptococci. Overlapping synthetic peptides that span the conserved carboxylterminal segment of the M-5 protein were constructed and used to immunise a panel of H-2 congenic mice. Proliferative T-cell epitopes were identified and, in many cases, mice immunised with these peptides produced high titer antibodies to the same peptides indicating that these proliferative epitopes could also stimulate Th cells. Peptide-specific T cells and antisera were tested for their reactivity with porcine heart myosin, tropomyosin, human heart myosin synthetic peptides, mitral heart valve from patient with rheumatic heart disease, and extracts of human pericardial and atrial heart tissue. Although lymph node cells from different strains of mice showed no cross-reaction in several assays, peptide-specific antisera reacted to immunoblotted porcine myosin and to an tmmunoblotted extract of human atrial heart tissue. However, two conserved peptides, LRRDLDASREAKKQVEKALE and KLTEKEKAELQAKLEAEAKA, stimulated peptide-specific antibodies in BIO.BR and B10,D2 mice respectively, which reacted minimally if at all with human atrial heart tissue extract. Furthermore, antisera to the former peptide (referred to as peptide 145), in a bactericidal assay involving human neutrophils opsonize different isolates of group A streptococci (expressing between them five different M serotypes) taken from Aboriginal and Thai patients with pharyngitis or acute rheumatic fever. This provided evidence that conserved epitopes can be the targets of bactericidal antibodies. In human subjects, T cells from Caucasians with past histories of rheumatic fever and other heart disease responded more frequently to several peptides than normal subjects. In Thai subjects, the responses were frequent in normal subjects. T cell lines and clones were studied. Two cell lines from a normal Caucasian generated against two M peptides (including peptide 145) responded to myosin peptides, human heart tissue proteins, and porcine heart myosin, one cell line from a Thai rheumatic heart disease patient generated against another peptide responded to myosin peptides but not human heart tissue proteins or porcine heart myosin. Furthermore, peptide 145 is immunodominant for human antibodies being recognised by >90% of adult Aboriginal and Thai sera tested, as determined by ELISA. Using peptide competition it was shown that specific antibodies present in Aboriginal sera could opsonize group A streptococci. The minimal epitope within the peptide is conformational and cannot be represented by spanning 8-mer or 12-mer synthetic peptides. Thus T cell epitopes in the M protein may be human autoepitopes and may play an important role in rheumatic heart disease. However, since auto-reactive T cell lines could be generated from normal donors, other factors must also be involved in the aetiology of rheumatic heart disease.In conclusion, The results in this thesis have shown that conserved region of M protein contain both T-cell and B-cell helper epitopes which will be a target of group A streptococcal vaccine development. The conserved region of M protein also contain T-cell and B-cell cross-reactive epitopes. This may be an evidence for pathogenesis of rheumatic heart development.