Wendy A. Hayman

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 Australias 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.

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.

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.

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology.

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid‐core peptide technology was employed. Lipid‐core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1→LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side‐chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side‐chains increased immunogenicity. An increase in immunogenicity (enzyme‐linked immunosorbent assay (ELISA) titres) of up to 100‐fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freunds adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins.

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.

Intranasal immunisation of mice with a streptococcal peptide-based vaccine.

The comparative roles of systemic and local responses in immune protection after group A streptococcal (GAS) infection are not fully understood. After parenteral immunisation of mice with M protein type-specific and conserved region peptides, there is an induction of serum bactericidal antibodies (Abs). In humans, type-specific immunity to GAS infection is long lasting, and Abs to the conserved region of M protein increase with age and immune status. Opsonic serum Abs may only form part of the protective response as there is also evidence for a protective role for secretory IgA (sIgA). Passive transfer of M protein-specific human sIgA significantly inhibited streptococcal infection in mice1. Intranasal (i.n.) immunisation with conserved peptides linked to cholera toxin subunit B (CTB) resulted in a significant reduction in pharyngeal colonisation of mice following homologous and heterologous GAS challenge2–4. These data suggest that mucosal protective responses may be directed to non-type specific regions of the M protein, however there was a failure to demonstrate the presence of M protein peptide-specific sIgA. We are further investigating the role of an M protein peptide-based mucosal vaccine to prevent infection and rheumatic fever. Mucosal immunisation has the advantage of inducing immune responses effective at preventing attachment/colonisation in the throat (a site of natural infection), and then triggering systemic immunity important for eliminating any invading bacteria. Work in our laboratory has focussed on a 20 amino acid epitope, p145 (LRRDLDASREAKKQVEKALE) within the conserved C-repeat region of the M protein. Abs to p145 raised after subcutaneous (s.c.) immunisation of mice could effectively opsonise and direct killing of a number of field isolates and reference strains5.