What type of vaccine is streptococcus pyogenes capsule

The amino-terminus of the M-protein is considered to be hypervariable, and used to define the more than different GAS emm-types [ 10 ].

Natural and vaccine-induced antibodies to this region are bactericidal, but typically only confer emm-type specific protection [ 11 — 13 ]. The presence of epitopes in the B-repeat region of the protein associated with autoimmune sequelae [ 14 ] preclude its use in any vaccine candidate.

A Schematic diagram of the M-protein. The location of J14 i variant peptide sequences within each of the CRUs are shown as black boxes. The figure is not drawn to scale.

B Schematic of SV1. The location and identity of each J14 i sequence is shown. The CRR of most M-proteins contains 3 repeat units that are similar, but not identical [ 20 , 21 ].

The variant sequences present in individual C-repeat units CRU are named on the basis of differences in an internal amino acid sequence that corresponding to the J8 i peptide or overlapping J14 i peptide sequences [ 21 , 22 ]. The J14 i variant sequences found in these repeat units are generally conserved within an emm-type, but vary between emm-types.

Currently 76 different J14 i types have been described. When flanked by amino acid sequence required for maintenance of alpha-helical structure, and linked to carrier molecule such as diphtheria toxoid, the prototype J8 i and J14 i peptide sequences have been shown to induce antibody responses that are bactericidal and protect mice from GAS challenge [ 16 , 17 ]. Our approach to GAS vaccine development has been to identify common J14 i variant sequences present in different C-repeat units CRUs and link them into a single recombinant construct Fig 1 [ 19 ].

SV1 contains five such sequences J14 i. Each one of the J14 i variant peptides present in SV1 consists of 14 amino acids. Consequently, SV1 maintains an alpha-helical structure without the need for additional flanking sequence and SV1 is also immunogenic in mice. Anti-SV1 antibodies also bind to the surface of three GAS emm-types emm 1, emm 97 and emm 65 , and are bactericidal towards the two GAS emm-type emm 97 and emm 65 strains tested.

The aim of the current study is to assess the emm-coverage afforded by SV1 and the immuno-safety of this vaccine. Furthermore, SV1 antibodies recognize J14 i variants not present in the vaccine construct, potentially extending vaccine coverage to emm-types that lack representation in the SV1 vaccine construct. Finally we also demonstrate that SV1 immunization does not induce cross-reactive immune responses as determined using a Lewis Rat model of valvulitis.

Together these results suggest that SV1 has significant potential as a safe and universal GAS vaccine candidate.

The GAS strains used in this study Table 1 have all previously been described [ 20 , 24 , 25 ]. They represent 9 different emm-types, and one emm-negative strain. The strains also represent each of the major clusters proposed as part of the new emm-types clustering system [ 20 ].

Recombinant proteins were expressed in Escherichia coli and purified using nickel affinity chromatography. Blood was collected prior to each immunization and 7 days after the final immunization. Sera from the final bleed was used in all assays reported here. Peptide solutions were assessed at 0. The amount of TFE required to observe alpha-helical secondary structure is based on the appearance of a peak at nm. The absorbance nm was then measured.

Antibody titers were defined as the reciprocal of the highest dilution of samples that yielded an optical density at nm of more than 3 standard deviations above the mean optical density of pre-immune sera. Immunofluorescent microscopy was carried out essentially as previously described [ 29 ].

To prevent non-specific Fc-IgG binding by streptococcal surface proteins the slides were exposed to a second blocking step using non-specific human polyclonal IgG AbD Serotec was undertaken. The slides were mounted in Prolong Gold Life Technologies and viewed with a x objective lens using a nm laser with absorbance of nm on an Olympus Fluoview FV Confocal Laser Scanning microscope.

Following 60 min incubation, the membrane was washed, covered with chemiluminescent substrate ECL Western Blotting Detection Reagent, Amersham following manufactures instructions and exposed to standard X-ray film.

The immuno-safety of SV1 was evaluated in a Lewis rat model of autoimmune valvulitis [ 30 , 31 ]. Eight to twelve week-old female Lewis rats were subcutaneously immunized on day 0 in the hock of the hind left foot with 0. Negative control rats were immunized with PBS in adjuvant. On days 1 and 3, the rats were intraperitoneally injected with 0. Antigen boosts consisting of 0. All animals were euthanased on day 21 by CO2 asphyxiation in a lethal chamber, followed by cervical dislocation.

Blood, hearts and spleens were then collected. Mononuclear cells were aseptically isolated from spleens and T cell proliferation assays performed.

The cells were pulsed with 0. Proliferative responses are reported as the change in CPM between stimulated and unstimulated cells. Histological studies on cardiac tissue were carried out on rat hearts fixed in neutral buffered formalin and embedded in paraffin. FACS was used to assess the binding of anti-sera to the surface of three GAS isolates that differed in their capsule expression levels [ 19 ]. GAS is an emm 1 clinical isolate associated with invasive disease outbreaks [ 32 ].

Statistical significant differences in MFI between PBS and experimental groups was subsequently assessed using by t-test.

The selection of the J14 i variants present in SV1 was originally made after the examination of genes representing 77 different emm-types that were present in the Genbank database [ 19 ]. A subsequent study of emm-gene sequences of greater than globally distributed GAS isolates provided the opportunity to expand the analysis of the coverage of J14 i variants represented in SV1 to a larger dataset that include unique emm-genes [ 23 ].

As the earlier study showed that the C-terminal amino acid sequences to be highly conserved within an emm-type, and to avoid bias due to over-representation, individual representatives of each emm-type were selected for the current analysis. Of the unique emm-genes, all but five emm, emm, emm, emm and emm contained at least one of the J14 i variants represent in SV1 S1 Table.

J14 i. Finally, individual CRUs were present within the M-proteins analyzed. Of these, possessed a J14 i variant present in SV1. To show that anti-SV1 antibodies did actually recognise and bind to the individual J14 i variants represented in the vaccine, sera collected after final immunisation of mice was used in ELISA against a panel of J14 i variant peptide sequences Fig 2.

The panel included the variants present in SV1 and five additional J14 i sequences. These latter peptides were included to assess the capacity of SV1 antibodies to bind other J14 i sequences and were selected for inclusion on the basis of their ubiquity J14 i. The mean titers for the five SV1 represented variants ranged from 3.

For the peptides not present in SV1, the highest titers were observed using J14 i. There were no significant differences between the mean titers observed for these two peptides, and those observed with any of the peptides presents in SV1 as determined using by ANOVA and Kruskal Wallace non-parametric test. Titers against the remaining three other peptides J14 i.

Together these results demonstrate anti-SV1 antibodies are capable of recognizing multiple J14 i variants, increasing targets for SV1 antibodies on the bacterial surface, and potentially increasing the emm-type coverage afforded by SV1. Black bars represent mean titers observed using anti-SV1 antisera. Open bars represent mean titres observed using anti-PBS antisera. To determine which variants present of SV1 contribute to cross-recognition, antisera were raised against J14 i peptides.

As individual J14 i peptides are poorly immunogenic, the J14 i peptides were resynthesized with GCN4 flanking amino acid sequences, designed to maintain the alpha-helical conformation of the parental M-protein, and conjugated to diphtheria toxoid prior to immunization of mice. Sera from individual mice in each group were then pooled and used in ELISAs against the panel of 10 J14 i variant peptides. For these assays, a titer of was chosen as an arbitrary cut-off value representing low or no binding between sera and peptide Table 4.

As expected, antibody titers for individual sera were highest when used against the corresponding J14 i peptide sequence as capture antigen. Anti-J14 i. Titers for anti-J14 i. Sera raised to J14 i. Despite showing low titers against J14 i. Collectively, nine of the ten peptides examined were recognized by at least one of these peptide antisera. The differences in peptides recognized by the different anti-sera underscore the utility of incorporating multiple J14 i variants in a single construct.

To identify factors that may contribute to the cross-recognition, the percentage identity between J14 i peptides was determined Table 5 , and compared to the corresponding ELISA titer Fig 3. This analysis has shown that for sera tested, there was a correlation between titer and relative identity between peptides. Percent identity is plotted on the x-axis. Corresponding titers are plotted on the y-axis. Linear regression revealed that a significant correlation between these two variables exists for each antisera.

The dotted horizontal line shows the cut-off used as the titre used to differentiate between low or non-significant binding, and significant binding. The data above demonstrate that the design strategy for SV1 has resulted in a recombinant construct that is immunogenic and evokes antibody responses in the presence of a human approved adjuvant that theoretically should be capable of binding to the CRR of multiple M-proteins.

To confirm this we subsequently conducted Western blots against eight M-proteins representing different emm-types and emm-type clusters. Binding was observed in all instances Fig 4. The emm-type and emm -cluster assignment are shown at the top of the figure. Images are shown as overlays of bright field and fluorescent images. No fluorescence was observed when PBS sera was used in the same assays data not shown. The presence of cross-reactive epitopes within the M-protein has been a major hurdle for all M-protein vaccine development programs.

The ability to demonstrate that immune responses raised against vaccine candidates do not induce immune responses that cross-react with human proteins and tissues is an important step before vaccines can proceed to human clinical trials. Here we first used Western blots using porcine cardiac myosin and skeletal muscle tropomyosin to show that antibodies from SV1 immunised mice failed to react with these proteins S1 Fig.

The rat autoimmune valvulitis model is the only animal model available that adequately mirrors the pathophysiological futures of RHD in humans. Many FBPs also inhibit activity of the complement system in a fibronectin-independent manner.

For example, M1 protein and FbaA bind to factor H and related proteins which negatively regulate complement activity Smeesters, McMillan and Sriprakash ; Ma et al. Neutralising antibodies that recognise FBPs could potentially block these interactions and increase susceptibility to immune clearance. Due to their localisation at the cell surface and expression in multiple strains, FBPs are potential vaccine candidates and have been investigated in pre-clinical challenge models Table 1.

Vaccination of mice with type I FBPs has shown varying levels of protection. Sfb1 provided some long-term protection against intranasal GAS challenge Schulze et al. Antibodies to FbaA are bactericidal in vitro and vaccination is protective against intraperitoneal challenge in a mouse model Terao et al. Fbp54 is highly immunogenic in humans and partially protective against oral, intranasal and subcutaneous challenge with multiple serotypes in mice. Kawabata et al. Shr is highly conserved amongst strains Eichenbaum and immunisation protected against multiple strains in both invasive and nasopharyngeal mouse challenge models Huang et al.

GAPDH is a ubiquitous glycolytic enzyme that is also found bound to the cell surface in GAS where it has multiple functions including fibronectin binding Jin et al. However, due to the expression of GAPDH in humans as well as non-GAS bacteria there is theoretical potential for off-target effects and autoimmunity if this were to be a vaccine component. These studies present mixed results for the FBP family as a whole, yet indicate that some may be worth investigating further as vaccine candidates.

Conserved proteins that protect from multiple routes of infection, such as FBP54 or Shr, perhaps show the greatest potential for a broadly effective vaccine.

To date, the structures of very few GAS adhesins have been solved Fig. In addition to the pilus components FctA Pointon et al. In contrast, M1 and Sfb1 residues and residues respectively are estimated to extend less than nm from the cell Fig. Due to this large difference in size and the repetitive nature of the pilus backbone, epitopes on the T-antigen are potentially more available to the immune system and less likely to be masked by GAS-bound ECM components. After decades of limited research, GAS vaccine development is now an active field with numerous candidates being investigated.

Adhesin-based vaccines are attractive targets, and in general pre-clinical results have been promising. However, questions regarding strain coverage and population-based efficacy remain that may partly be answered in forthcoming clinical trials.

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Introduction Streptococcus pyogenes , also known as Group A Streptococcus GAS , is a human pathogen that is estimated to cause over , deaths annually 1. Results Cloning and expression of TeeVax1 Previous bioinformatic analysis of two-domain T-antigens revealed a highly conserved core decorated by surface variation along the full length of the protein, with no apparent dominant region Figure 1. Full size image. Figure 2. Figure 3.

Figure 4. Figure 5. Discussion GAS is one of the top ten causes of death by an infectious agent worldwide and the need for a GAS vaccine is clear. Bacteria and cell culture conditions GAS strains used in this study are listed in Supplementary Table 1. Cloning and protein expression The tee or emm1 genes were amplified from genomic DNA between the predicted signal peptide cleavage site and sortase motif by PCR using iProof high-fidelity polymerase Bio-rad with primers listed in Supplementary Table 2.

Statistical analysis All statistical analysis was performed using GraphPad Prism software. References 1.