A conformationally constrained, minimally conserved peptide from the M protein of group A streptococcus (GAS) has been defined. It consists of 12 amino acids from the C-repeat region within a non-M protein helix-forming sequence and is referred to as "J8." Here, we investigate the immunogenicity of a J8-diphtheria toxoid (DT) conjugate adjuvanted with the human-compatible adjuvants, SBAS2 and alum, and demonstrate that it is capable of inducing opsonic antibodies and can protect outbred mice from virulent challenge. In a range of experiments, protection correlated with the titer of J8-specific antibodies and not with the induction of J8-specific T cells. However, DT-specific antibodies (as well as J8-specific antibodies) were shown to stain the surface of fixed GAS and to be capable of opsonizing live organisms. DT may be an ideal carrier protein for J8 and other GAS peptides for GAS vaccines.

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.

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Rheumatic fever (RF) follows a throat infection with different M-serotypes of beta-hemolytic group A streptococci (GAS) and can affect different tissues, predominantly the heart. It is thought to be an autoimmune illness. Although histological examination of affected heart shows an infiltrate consisting mainly of T cells, antigens or epitopes that could be putative targets of autoimmune T cells have not been identified. We have examined the T cell response to the conserved C-terminal region of the M protein--a streptococcal surface coiled-coil protein which is the target of opsonic antibodies and antibodies which cross-react with human heart tissue. Australian Aborigine, Caucasian and Thai patients, controls and mice were studied to define regions of the protein immunogenic for T cells, and T cell lines and clones were tested for cross-reactivity to myosin as well as an extract of RF-diseased mitral heart valve. Murine (B10, B10.D2, B10.BR) M peptide-specific T cells were often cross-reactive for other M peptides but did not cross-react with human heart antigens. Patients with RF or other heart diseases, or control subjects exposed more commonly to GAS were more likely to have T cell responses to the M protein, with many regions of the C-terminus being recognized. T cell lines and a clone specific for different M peptides were generated from five donors. Cross-reactivity could be shown between different M peptides, but unlike murine M peptide-specific T cells three of the human T cell lines reacted strongly to peptides representing homologous regions of cardiac and skeletal muscle myosins, and two of these lines also responded to porcine myosin and an extract of human rheumatic mitral valve. However, these last two lines were derived from a normal donor without history of RF or other heart disease. Our data demonstrate that regions of the M protein, including regions that are being considered as subunit vaccines, have the potential to stimulate pre-existing heart cross-reactive T cells, but that the ability of such T cells to cross-react (as measured in vitro) is not in itself sufficient to lead to disease.