New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population.

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.

Human antibodies to the conserved region of the M protein: opsonization of heterologous strains of group A streptococci.

A 20-mer peptide (p145) in the carboxyl-terminal region of the M protein of group A streptococci (GAS) has previously been defined as the target of bactericidal antibodies. Sequence analysis of seven field isolates from indigenous Australians living in an area highly endemic for GAS and five laboratory reference strains (encompassing nine unique serotypes plus three nontypeables) demonstrates that this region is highly conserved (sequence identity ranging from 65 to 95%) with six of the 12 sequences being identical to p145. Most of the sequence dissimilarity is contained within the last seven amino acids of p145. Competitive ELISA demonstrates that human antibodies specific for p145 cannot discriminate between p145 and synthetic peptides representing four from four of the variant sequences tested. Ig purified from endemic sera was able to opsonize each of the GAS isolates and free p145 as well as a peptide expressing a minimal conformational epitope within p145 (requiring amino acids between positions 2 and 13 of p145), but not an irrelevant peptide, were able to partially or completely inhibit opsonization of all isolates and reference strains. Thus adult endemic sera contain antibodies which are bactericidal for multiple GAS serotypes and which are specific for a sequence of 12 amino acids contained within the p145 region of the M protein.

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.

Mapping a conserved conformational epitope from the M protein of group A streptococci.

The carboxyl terminus of the M protein of group A streptococci (GAS) is highly conserved and contains epitopes that have been shown to induce opsonic antibodies and protection against GAS infection. This region of the protein can also stimulate T cells, which can react in vitro with heart antigens. Since different segments of the carboxyl terminus may be involved in immunity to GAS and in the pathogenesis of autoimmune disease (rheumatic heart disease), it is important to precisely define critical epitopes. However, the M protein is known to be a coiled coil, and a critical immunodominant antibody-binding epitope within this region (peptide 145, a 20-mer with the sequence LRRDLDASREAKK-QVEKALE) is shown here to be conformational. Thus, small synthetic overlapping peptides of 8-12 amino acids in length that span peptide 145 (p145) were unable to capture antibodies present in p145-immune mouse sera or in endemic human sera, even though antibodies raised to these small peptides coupled to diphtheria toxoid could bind the smaller peptides and, in some cases, p145. A series of mutated peptides in which every residue of p145 was sequentially altered also failed to identify critical residues for antibody binding. We thus devised a strategy to produce chimeric peptides in which small peptides copying the M protein sequence were displayed within a larger 28-mer peptide derived from the sequence of the GCN4 leucine zipper DNA binding protein of yeast. A 12-amino-acid window of the p145 sequence was inserted into the GCN4 peptide in such a way as to preserve any potential helical structure. The window was moved along one residue at a time to give a series of peptides representing p145. Circular dichroism demonstrated that these larger chimeric peptides and p145, but not a shorter 12-mer peptide, displayed alpha-helical potential in 50% trifluoroethanol. Certain chimeric peptides efficiently captured antibodies specific for p145 and thus enabled us to map the minimal antibody-binding sequence. RRDLDASREAKK, referred to as J(1)2. The chimeric peptide containing this sequence, referred to as J2, was able to inhibit opsonization of GAS by human antisera containing anti-peptide 145 antibodies. The T-cell response from p145-immunized responder B10.BR mice to J2 and J(I)2 was much lower than the response to p145 and mapped to a different peptide.

Functional analysis of IgA antibodies specific for a conserved epitope within the M protein of group A streptococci from Australian Aboriginal endemic communities.

The mucosa is one of the initial sites of group A streptococcal (GAS) infection and salivary IgA (sIgA) is thought to be critical to immunity. However, the target epitopes of sIgA and the function of sIgA in GAS immunity, in particular the role of accessory cells and complement, is largely unknown. We studied the aquisition and the function of sIgA specific for a conserved region epitope, p145 (sequence: LRRDLDASREAKKQVEKALE) of the M protein. Peptide 145-specific sIgA is highly prevalent within an Aboriginal population living in an area endemic for GAS and acquisition of p145-specific sIgA increases with age, consistent with a role for such antibodies in immunity to GAS. Human sIgA and IgG specific for p145 were affinity purified and shown to opsonize M5 GAS in vitro. Opsonization could be specifically inhibited by the addition of free p145 to the antibodies during assay. Opsonization of GAS was totally dependent on the presence of both complement and polymorphonuclear leukocytes, and, moreover, affinity-purified p145-specific sIgA was shown to fix complement in the presence of M5 GAS. These data show that mucosal IgA to this conserved region peptide within the M protein has an important role in human immunity against GAS and may be useful in a broad-based cross-protective anti-streptococcal vaccine.

Opsonic human antibodies from an endemic population specific for a conserved epitope on the M protein of group A streptococci.

This study demonstrates the presence of epitope-specific opsonic human antibodies in a population living in an area endemic for group A streptococci (GAS) infection. Antibodies recognizing a conserved C-terminal region epitope (p145, sequence in single letter amino acids: LRRDLDASREAKKQVEKALE) of the M protein of GAS were isolated from human patients by affinity chromatography and were shown to be of the immunoglobulin G1 (IgG1) and IgG3 subclasses. These antibodies could reduce the number of colonies of serotype 5 GAS in an in vitro opsonization assay by 71-92%, compared with an equal amount of IgG from control adult donors living in non-endemic areas and without antibodies to p145. Addition of the peptide, p145, completely inhibited this opsonization. Indirect immunofluorescence showed that p145-specific antibodies were capable of binding to the surface of M5 GAS whereas control IgG did not. Using chimeric peptides, which contain overlapping segments of p145, each 12 amino acids in length, inserted into a known helical peptide derived from the DNA binding protein of yeast, GCN4, we have been able to further define two minimal regions within p145, referred to as pJ2 and pJ7. These peptides, pJ2 and pJ7, were able to inhibit opsonization by p145 specific antibodies. Finally, we have observed an association between the age-related development of immunity to GAS and the acquisition of antibodies to the conserved epitope, p145, raising the possibility of using this epitope as a target in a prophylactic vaccine administered during early childhood.