Evelyn R. Brandt

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.

Towards a vaccine for rheumatic fever: identification of a conserved target epitope on M protein of group A streptococci

Rheumatic fever and rheumatic heart disease remain very common in developing countries, and a vaccine to protect against these disorders would have a great impact on public health. A vaccine must target the M protein of group A streptococci (Streptococcus pyogenes), but until lately immunity was thought to be strain-specific and dependent on antibodies to the variable serotype-specific regions of the protein. Experiments in animals have suggested the conserved region of the M protein as a possible alternative target for protective antibodies. We constructed a 20-aminoacid peptide (peptide 145) within the conserved region of the carboxyl terminus of the protein. In mice the peptide induced serum antibodies that could opsonise reference type 5 streptococci. By enzyme-linked immunosorbent assay, positive responses to peptide 145 were obtained with serum from 77 (90%) of 86 Aboriginal subjects and 135 (81%) of 167 Thai subjects living in areas with high exposure to streptococci. Only 10 (14%) of 71 Caucasian subjects with low exposure to streptococci showed positive responses. There was no difference in the proportion positive between subjects with rheumatic heart disease and control groups (other or no heart disease). Antibodies to peptide 145 were able to opsonise isolates of streptococci from Aboriginal and Thai subjects with acute rheumatic fever as well as reference strains. This highly conserved part of the M protein may be a suitable target for vaccines to prevent streptococcal infections and their sequelae.

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.

Protective and Nonprotective Epitopes from Amino Termini of M Proteins from Australian Aboriginal Isolates and Reference Strains of Group A Streptococci

ABSTRACT The M protein is the primary vaccine candidate to prevent group A streptococcal (GAS) infection and the subsequent development of rheumatic fever (RF). However, the large number of serotypes have made it difficult to design a vaccine against all strains. We have taken an approach of identifying amino-terminal M protein epitopes from GAS isolates that are highly prevalent in GAS-endemic populations within the Northern Territory (NT) of Australia. Australian Aboriginals in the NT experience the highest incidence of RF worldwide. To develop a vaccine for this population, 39 peptides were synthesized, representing the amino-terminal region of the M protein from endemic GAS. Mice immunized with these peptides covalently linked to tetanus toxoid and emulsified in complete Freunds adjuvant raised high-titer antibodies. Over half of these sera reduced bacterial colony counts by >80% against the homologous isolate of GAS. Seven of the peptide antisera also cross-reacted with at least three other heterologous peptides by enzyme-linked immunosorbent assay. Antiserum to one peptide, BSA101–28, could recognize six other peptides, and five of these peptides could inhibit opsonization mediated by BSA101–28 antiserum. Cross-opsonization studies showed that six of these sera could opsonize at least one heterologous isolate of GAS. These data reveal vaccine candidates specific to a GAS-endemic area and show the potential of some to cross-opsonize multiple isolates of GAS. This information will be critical when considering which epitopes may be useful in a multiepitope vaccine to prevent GAS infection.

Vaccine strategies to prevent rheumatic fever

Group A streptococci (GAS) are responsible for numerous human illnesses, ranging from pharyngitis to severe invasive infections, such as necrotizing fascitis and toxic shock syndrome to the postinfectious sequelae, acute rheumatic fever (ARF), and glomerulonephritis. To date, to develop a vaccine, studies have focused on the M protein. However, designing a vaccine to prevent GAS infection based on this molecule has been hampered by the vast number of M protein serotypes and the possibility that it may induce potentially harmful autoimmune reactions. In this article, the authors discuss recent approaches to overcoming the problems of an M proteinbased vaccine. In addition, recent studies identifying the protective properties of other streptococcal antigens and their potential as vaccine candidates are discussed.