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Streptococcus pyogenes, a leading human pathogen, is responsible for a wide range of diseases, including skin and soft tissue infections and severe invasive diseases. S. pyogenes produces a large arsenal of virulence factors, including several immune evasion factors. We have identified an open reading frame (spy0136) in the S. pyogenes SF370 genome encoding a protein of unknown function. Using recombinant Spy0136 in a pull-down assay with human plasma and ELISA, we have identified four complement proteins (C1r, C1s, C3, and C5) as binding partners. Treatment of the complement proteins with PNGase F abrogated binding to C1s, C3, and C5, indicating glycan-dependent interactions. rSpy0136 inhibited complement-mediated hemolysis and interfered with all three complement pathways in a Wieslab complement assay. Furthermore, rSpy0136 inhibited deposition of the C3b opsonin and the membrane attack complex (MAC) on the surface of S. pyogenes. We therefore named the previously unknown protein ‘complement evasion factor’ (CEF). An S. pyogenes Δspy0136/cef deletion mutant showed decreased virulence in an in-vitro whole blood killing assay and a Galleria mellonella (wax moth) infection model. Furthermore, an L. lactis spy0136/cef gain-of-function mutant showed increased survival during growth in whole human blood. Analysis of serum samples from patients with invasive S. pyogenes revealed Spy0136/CEF sero-conversion indicating expression during disease. In summary, we have identified a novel S. pyogenes immune evasion factor that binds to several complement proteins to interfere with complement function. This is the first example of a S. pyogenes virulence factor binding to several different target proteins via glycan-dependent interactions.

Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating “knee-joint” like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.

Mycobacterium tuberculosis (Mtb) remains a global epidemic despite the widespread use of Bacillus Calmette-Guerin (BCG). Consequently, novel vaccines are required to facilitate a reduction in Mtb morbidity and mortality. PilVax is a peptide delivery strategy for the generation of highly specific mucosal immune responses and is based on the food-grade bacterium Lactococcus lactis that is used to express selected peptides engineered within the Streptococcus pyogenes M1T1 pilus, allowing for peptide amplification, stabilization and enhanced immunogenicity. In the present study, the dominant T-cell epitope from the Mtb protein Ag85B was genetically engineered into the pilus backbone subunit and expressed on the surface of L. lactis. Western blot and flow cytometry confirmed formation of pilus containing the peptide DNA sequence. B-cell responses in intranasally vaccinated mice were analyzed by ELISA while T-cell responses were analyzed by flow cytometry. Serum titers of peptide-specific immunoglobulin (Ig) G and IgA were detected, confirming that vaccination produced antibodies against the cognate peptide. Peptide-specific IgA was also detected across several mucosal sites sampled. Peptide-specific CD4+ T cells were detected at levels similar to those of mice immunized with BCG. PilVax immunization resulted in an unexpected increase in the numbers of CD3+ CD4- CD8- [double negative (DN)] T cells in the lungs of vaccinated mice. Analysis of cytokine production following stimulation with the cognate peptide showed the major cytokine producing cells to be CD4+ T cells and DN T cells. This study provides insight into the antibody and peptide-specific cellular immune responses generated by PilVax vaccination and demonstrates the suitability of this vaccine for conducting a protection study.

The group A Streptococcus (GAS) pilus is a long, flexible, hair-like structure anchored to the cell surface that facilitates the adherence of GAS to host cells, thus playing a critical role in initiating infections. Because of its important role in GAS virulence, the pilus has become an attractive target for vaccine development. While current research mainly focuses on pilus function and its potential as a vaccine component, there is a lack of knowledge on how the host immune system recognizes and responds to this abundant surface structure. Here we show that both assembled GAS pili and individual pilus proteins induce a potent release of the proinflammatory cytokines tumor necrosis factor and interleukin-8. We further show that the surface-exposed backbone pilin and ancillary pilin 1 subunits are Toll-like receptor 2 (TLR2) agonists. Using reporter cell lines coexpressing human TLR2 in combination with either TLR1 or TLR6, we determined that activation was mediated by the TLR2/TLR6 heterodimer. Finally, we used solid-phase and flow cytometry binding assays to illustrate a direct interaction between the pilus subunits and TLR2. These results provide further support for the suitability of the pilus as a vaccine component and opens potential avenues for using GAS pili as an adjuvant or immune-modulation agent.

Group A Streptococcus (GAS) is a major human pathogen responsible for superficial infections through to life-threatening invasive disease and the autoimmune sequelae acute rheumatic fever (ARF). Despite a significant global economic and health burden, there is no licensed vaccine available to prevent GAS disease. Several pre-clinical vaccines that target conserved GAS antigens are in development. Assays that measure antigen-specific antibodies are essential for vaccine research. The aim of this study was to develop a multiplex beadbased immunoassay that can detect and quantify antibody responses to multiple GAS antigen targets in small volume blood samples. This builds on our existing triplex assay comprised of antigens used in clinical serology for the diagnosis of ARF (SLO, DNase B and SpnA). Five additional conserved putative GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD and the Group A carbohydrate), were coupled to spectrally unique beads to form an 8-plex antigen panel. After optimisation of the assay protocol, standard curves were generated, and assessments of assay specificity, precision and reproducibility were conducted. A broad range of antibody (IgG) titres were able to be quickly and accurately quantified from a single serum dilution. Assay utility was assessed using a panel of 62 clinical samples including serum from adults with GAS bacteraemia and children with ARF. Circulating IgG to all eight antigens was elevated in patients with GAS disease (n = 23) compared to age-matched controls (n = 39) (P 0.05). The feasibility of using dried blood samples to quantify antigen-specific IgG was also demonstrated. In summary, a robust and reproducible 8-plex assay has been developed that simultaneously quantifies IgG antibodies to GAS vaccine and diagnostic antigens.

Streptococcal serology is a cornerstone in the diagnosis of acute rheumatic fever (ARF), a postinfectious sequela associated with group A Streptococcus infection. Current tests that measure anti-streptolysin O (ASO) and anti-DNaseB (ADB) titers require parallel processing, with their predictive value limited by the low rate of decay in antibody response. Accordingly, our objective was to develop and assess the diagnostic potential of a triplex bead-based assay, which simultaneously quantifies ASO and ADB together with titers for a third antigen, SpnA. Our previous cytometric bead assay was transferred to the clinically appropriate Luminex platform by coupling streptolysin O, DNaseB, and SpnA to spectrally unique magnetic beads. Sera from more than 350 subjects, including 97 ARF patients, were used to validate the assay and explore immunokinetics. Operating parameters demonstrate that the triplex assay produces accurate and reproducible antibody titers which, for ASO and ADB, are highly correlative with existing assay methodology. When ARF patients were stratified by time (days following hospital admission), there was no difference in ASO and ADB between

The TCR Vβ repertoire from patients with recurrent tonsillitis and/or tonsillar hyperplasia was examined to determine whether the TCR Vβ composition is suggestive of local superantigen activity and if so, whether it is associated with the presence of superantigen producing bacteria. Tonsil specimens were cultured aerobically to allow identification and isolation of the bacterial pathogens Staphylococcus aureus and Group A Streptococcus. TCR Vβ subset analysis of tonsil leucocytes was performed by flow cytometry. The superantigenic potential of tonsil S. aureus isolates was determined by multiplex PCR and a T-cell mitogenicity assay. Tonsils were collected from 40 patients who were predominantly pre-school-aged children undergoing surgery for either recurrent tonsillitis or tonsillar hyperplasia causing obstructive sleep apnoea. S. aureus was cultured from 23/40 and Group A Streptococcus from 5/40 patients. Both CD4+ and CD8+ TCR Vβ populations were skewed in 17/40 patients. Twelve of these had recurrent tonsillitis of whom 9 also harboured S. aureus. Characterisation of tonsillar S. aureus isolates revealed that many contained genes for one or more potent superantigens and detection of these genes was associated with in vitro mitogenic activity. Skewing of the tonsillar TCR Vβ repertoire was observed at high frequency and was most commonly associated with the presence of S. aureus. Many S. aureus isolates were mitogenic suggesting that they have a potential for local impact on the function of tonsil T cell populations. These results suggest the possibility that anti-staphylococcal antibiotics may be an effective treatment option for some patients.

Group A Streptococcus (GAS) often exists as an asymptomatic colonizer of the upper respiratory tract in humans. Unsurprisingly, a high proportion of symptomatic infections caused by GAS include pharyngitis. While not usually life-threatening, these infections cause significant morbidity and economic burden/loss of productivity, and can have downstream life-threatening autoimmune consequences. Modeling asymptomatic colonization in animals is, therefore, a useful tool to dissect host-bacteria interactions and to evaluate efficacy of vaccines aimed at reducing the burden of carriage. Here we describe a mouse model of nasopharyngeal colonization using nasal challenge of susceptible mice and the evaluation of subsequent bacterial burden.

Most pathogens initiate infection via the mucosa, therefore delivery of vaccines directly to the mucosa is likely to be advantageous for stimulating protective immunity at the site of entry. PilVax is a novel mucosal vaccine platform that harnesses Lactococcus lactis bacteria engineered to stably express multiple copies of vaccine peptide antigens within pili, hair-like structures which extend from the cell wall. This strategy elicited systemic and mucosal antibody responses to a model antigen after intranasal immunization, but has not been tested for its capacity to stimulate protective mucosal immunity. A well-characterized linear B-cell epitope, D3(22-33) , from the fibronectin-binding protein A of Staphylococcus aureus was successfully introduced into PilVax and delivered intranasally to mice. Specific antipeptide immunoglobulin (Ig) G and IgA antibodies were detected in the serum and respiratory mucosa of vaccinated mice. Responses to the major pilus backbone protein Spy0128 were also assessed; robust antibody responses to this antigen were generated both systemically and in the respiratory and intestinal mucosa. Mice were challenged intranasally with the mouse-adapted S. aureus JSNZ strain and the S. aureus load quantified 7 days after challenge. Unexpectedly, exposure to PilVax, irrespective of the presence of the peptide, resulted in a significant reduction in S. aureus load in both the intestine and nasal mucosa (both P < 0.05) when compared with unvaccinated control mice. The mechanism(s) of protection are unclear, but merit further investigation to determine whether PilVax is a suitable platform for delivery of vaccine candidate antigens to the mucosa.

Group A Streptococcus (GAS) (Streptococcus pyogenes) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines.

Galleria mellonella (greater wax moth or honeycomb moth) has been introduced as an alternative model to study microbial infections. G. mellonella larvae can be easily and inexpensively obtained in large numbers and are simple to use as they don't require special lab equipment. There are no ethical constraints and their short life cycle makes them ideal for large-scale studies. Although insects lack an adaptive immune response, their innate immune response shows remarkable similarities with the immune response in vertebrates. This review gives a current update of what is known about the immune system of G. mellonella and provides an extensive overview of how G. mellonella is used to study the virulence of Gram-positive and Gram-negative bacteria. In addition, the use of G. mellonella to evaluate the efficacy of antimicrobial agents and experimental phage therapy are also discussed. The review concludes with a critical assessment of the current limitatons of G. mellonella infection models.

To analyse the incidence, demographics and molecular epidemiology of invasive group A streptococcal (GAS) disease in New Zealand between 2002 and 2012.Using laboratory-based surveillance data, invasive GAS isolates were identified from the Institute of Environmental Science and Research, New Zealand. Hospitalization and mortality data were obtained from the New Zealand Ministry of Health. Molecular typing was performed by sequence analysis of the emm gene.The incidence of invasive GAS infections increased from 3.9 per 100,000 population in 2002 to 7.9 per 100,000 population (P 0.001) in 2012. The incidence was highest in the over 75-year age group, and in Pacific peoples. There was temporal variation in emm types associated with invasive GAS disease, with emm1 being the overall predominant emm type. The diversity of emm types varied significantly according to ethnicity. Overall, 59% of GAS isolates were theoretically covered by an experimental M-protein vaccine.Our study provides valuable data on the epidemiology of invasive GAS disease in New Zealand, and represents one of the few studies to assess such longitudinal data across an entire nation. The increase in invasive GAS disease is concerning, and reasons for this should be explored further.

Objectives Streptococcal serology provides evidence of prior Group A Streptococcus (GAS) exposure, crucial to the diagnosis of acute rheumatic fever (ARF) and post-streptococcal glomerulonephritis. However, current tests, which measure anti-streptolysin-O and anti-DNaseB antibodies, are limited by false positives in GAS endemic settings, and incompatible methodology requiring the two tests to be run in parallel. The objective was to improve streptococcal serology by combining the novel GAS antigen, SpnA, with streptolysin-O and DNaseB in a contemporary, bead-based immunoassay. Methods Recombinant streptolysin-O, DNAseB and SpnA were conjugated to polystyrene beads with unique fluorescence positions so antibody binding to all three antigens could be detected simultaneously by cytometric bead array. Multiplex assays were run on sera collected in three groups: ARF; ethnically matched healthy children; and healthy adults. Results The ability of the antigens to detect a previous GAS exposure in ARF was assessed using the 80th centile of the healthy children group as cut-off (upper limit of normal). SpnA had the highest sensitivity at 88%, compared with 75% for streptolysin-O and 56% for DNaseB. Conclusions SpnA has favorable immunokinetics for streptococcal serology, and can be combined with anti-streptolysin-O and anti-DNaseB in a multiplex format to improve efficiency and accuracy.

Group A streptococcus is a strict human pathogen that can cause a wide range of diseases, such as tonsillitis, impetigo, necrotizing fasciitis, toxic shock, and acute rheumatic fever. Modeling human diseases in animals is complicated, and rapid, simple, and cost-effective in vivo models of GAS infection are clearly lacking. Recently, the use of non-mammalian models to model human disease is starting to re-attract attention. Galleria mellonella larvae, also known as wax worms, have been investigated for modeling a number of bacterial pathogens, and have been shown to be a useful model to study pathogenesis of the M3 serotype of GAS. In this study we provide further evidence of the validity of the wax worm model by testing different GAS M-types, as well as investigating the effect of bacterial growth phase and incubation temperature on GAS virulence in this model. In contrast to previous studies, we show that the M-protein, among others, is an important virulence factor that can be effectively modeled in the wax worm. We also highlight the need for a more in-depth investigation of the effects of experimental design and wax worm supply before we can properly vindicate the wax worm model for studying GAS pathogenesis.

Acute rheumatic fever (ARF) is an autoimmune response to Group A Streptococcus (GAS) infection. Repeated GAS exposures are proposed to 'prime' the immune system for autoimmunity. This notion of immune-priming by multiple GAS infections was first postulated in the 1960s, but direct experimental evidence to support the hypothesis has been lacking. Here, we present novel methodology, based on antibody responses to GAS T-antigens, that enables previous GAS exposures to be mapped in patient sera. T-antigens are surface expressed, type specific antigens and GAS strains fall into 18 major clades or T-types. A panel of recombinant T-antigens was generated and immunoassays were performed in parallel with serum depletion experiments allowing type-specific T-antigen antibodies to be distinguished from cross-reactive antibodies. At least two distinct GAS exposures were detected in each of the ARF sera tested. Furthermore, no two sera had the same T-antigen reactivity profile suggesting that each patient was exposed to a unique series of GAS T-types prior to developing ARF. The methods have provided much-needed experimental evidence to substantiate the immune-priming hypothesis, and will facilitate further serological profiling studies that explore the multifaceted interactions between GAS and the host.

The complete set of currently known streptococcal superantigens (SAgs) was tested for efficacy to induce in vitro cytokine responses from 5 different blood donors using the Luminex 100 system. Cytokine responses varied significantly between individual SAgs and between donors with a mixed Th1/Th2 response in 3 donors and a predominantly Th1 or Th2 response in the other 2 donors. Our results suggest that streptococcal SAgs might be involved not only in acute streptococcal diseases but also in chronic inflammation and autoimmune diseases.

We report a nonfatal case of streptococcal toxic shock syndrome (STSS) caused by a Streptococcus pyogenes emm118 strain encoding a novel variant of streptococcal mitogenic exotoxin Z (SMEZ-34). This variant was responsible for the major mitogenic activity in the cell culture supernatant. Patient sera showed seroconversion toward SMEZ, implying a role for this toxin in STSS.

Streptococcus pyogenes is a human pathogen which causes a spectrum of diseases ranging from pharyngitis to rheumatic fever, necrotising fasciitis and toxic shock syndrome. Development of a vaccine for S. pyogenes has been confounded both by the diversity of the disease-causing serotypes and the spectre of inadvertently stimulating autoimmunity. The S. pyogenes nuclease A (SpnA) is a recently characterised virulence factor that is highly conserved across strains and expressed during human disease. Deletion of spnA from S. pyogenes results in reduced survival of bacteria in whole human blood and attenuated virulence in a mouse model of infection. Collectively these features suggest that SpnA has potential as a vaccine candidate for S. pyogenes. Mice vaccinated subcutaneously with single or multiple doses of recombinant SpnA emulsified in Incomplete Freund’s Adjuvant developed a robust and durable IgG response, including neutralising activity, to this protein. However, vaccination with rSpnA conferred no advantage in terms of lesion development, disease symptoms or colonisation levels after a sub-lethal subcutaneous challenge with S. pyogenes. Anti-SpnA serum IgG responses and neutralising activity were increased in response to challenge, indicating that SpnA is expressed in vivo. SpnA is unlikely to be a suitable antigen for a vaccine against S. pyogenes.

Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II β-chain in a zinc-dependent mode but not to the generic MHC class II α-chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DRβ–SPE-J–SPE-J–HLA-DRβ. SPE-I preferably stimulates T cells bearing the Vβ18.1 TCR, which is not targeted by any other known SAg. SPE-J almost exclusively stimulates Vβ2.1 T cells, a Vβ that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistinguishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C.

The frequencies of the newly identified streptococcal superantigen genes smez, spe-g, and spe-h were determined in a panel of 103 clinical isolates collected between 1976 and 1998 at various locations throughout New Zealand. smez and spe-g were found in every group A Streptococcus (GAS) isolate, suggesting a chromosomal location. The spe-h gene was found in only 24% of the GAS isolates and is probably located on a mobile DNA element. The smez gene displays extensive allelic variation and appears to be in linkage equilibrium with the M/emm type. 22 novel smez alleles were identified from 21 different M/emm types in addition to the already reported alleles smez and smez-2 with sequence identities between 94.5 and 99.9%. Three alleles are nonfunctional due to a single base pair deletion. The remaining 21 alleles encode distinct SMEZ variants. The mosaic structure of the smez gene suggests that this polymorphism has arisen from homologous recombination events rather than random point mutation. The recently resolved SMEZ-2 crystal structure shows that the polymorphic residues are mainly surface exposed and scattered over the entire protein. The allelic variation did not affect either Vβ specificity or potency, but did result in significant antigenic differences. Neutralizing antibody responses of individual human sera against different SMEZ variants varied significantly. 98% of sera completely neutralized SMEZ-1, but only 85% neutralized SMEZ-2, a very potent variant that has not yet been found in any New Zealand isolate. SMEZ-specific Vβ8 activity was found in culture supernatants of 66% of the GAS isolates, indicating a potential base for the development of a SMEZ targeting vaccine.

Superantigens have been implicated in a wide variety of human diseases. Yet, solid evidence for their role in pathogenesis is available only for Toxic Shock Syndrome and a few other conditions. This evidence is critically reviewed herein.

Three novel streptococcal superantigen genes (spe-g, spe-h, and spe-j) were identified from the Streptococcus pyogenes M1 genomic database at the University of Oklahoma. A fourth novel gene (smez-2) was isolated from the S. pyogenes strain 2035, based on sequence homology to the streptococcal mitogenic exotoxin z (smez) gene. SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ and streptococcal pyrogenic exotoxin (SPE)-C, whereas SPE-H is most similar to the staphylococcal toxins than to any other streptococcal toxin. Recombinant (r)SMEZ, rSMEZ-2, rSPE-G, and rSPE-H were mitogenic for human peripheral blood lymphocytes with half-maximal responses between 0.02 and 50 pg/ml (rSMEZ-2 and rSPE-H, respectively). SMEZ-2 is the most potent superantigen (SAg) discovered thus far. All toxins, except rSPE-G, were active on murine T cells, but with reduced potency. Binding to a human B-lymphoblastoid line was shown to be zinc dependent with high binding affinity of 15–65 nM. Evidence from modeled protein structures and competitive binding experiments suggest that high affinity binding of each toxin is to the major histocompatibility complex class II β chain. Competition for binding between toxins was varied and revealed overlapping but discrete binding to subsets of class II molecules in the hierarchical order (SMEZ, SPE-C) > SMEZ-2 > SPE-H > SPE-G. The most common targets for the novel SAgs were human Vβ2.1- and Vβ4-expressing T cells. This might reflect a specific role for this subset of Vβs in the immune defense of gram-positive bacteria.

Group A Streptococcus (GAS) infections represent a major public health burden in both developing and developed countries. In Australia and New Zealand GAS associated diseases are serious problems in Indigenous populations and a major cause of health inequality. Political recognition of these inequalities is providing impetus for strategies that reduce GAS disease and the development of a GAS vaccine now has governmental support in both Australia and New Zealand. Accordingly, an expert workshop was convened in March 2013 to consider available data on GAS vaccines. M-protein based vaccines constructed from the hyper-variable N-terminal region (30-valent vaccine) or the conserved C-repeat domain (J8 vaccine) were reviewed together with vaccine candidates identified using multi high-throughput approaches. Performing a comprehensive assessment of regional GAS strain epidemiology, defining the immune correlates of protection, and the establishment of clinical trial sites were identified as critical activities for a Trans-Tasman vaccine development programme.

Superantigens are key virulence factors in the immunopathogenesis of invasive disease caused by group A streptococcus. These protein exotoxins have also been associated with severe group C and group G streptococcal infections. A number of novel streptococcal superantigens have recently been described with some resulting confusion in their classification. In addition to clarifying the nomenclature of streptococcal superantigens and proposing guidelines for their categorization, this review summarizes the evidence supporting their involvement in various clinical diseases including acute rheumatic fever.

Summary: The bacterial superantigens are protein toxins that bind to major histocompatibility complex class II and T-cell receptor to stimulate large numbers of T cells. The majority are produced by the Gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes and are the causative agents in toxic shock syndrome, an acute disease caused by the sudden and massive release of T-cell cytokines into the blood stream. The structure and function of the superantigens has revealed a common architecture that is also shared by another group of staphylococcal virulence factors called the superantigen-like proteins (SSL). Together, this family of structurally related molecules highlights how a common pathogenic organism has employed a simple but adaptable protein to generate an armamentarium of potent defense molecules designed to target of the innate and adaptive immune response.

The exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes are prototype molecules for the larger family of superantigens (SAGs). This family now includes many structurally unrelated molecules of disparate origins reflecting a wide evolutionary convergence towards the common goal of

Eight different batches of intravenous immunoglobulin from 3 different manufacturers were tested for neutralizing activities against all currently known streptococcal superantigens. Statistically significant variation among different intravenous immunoglobulin preparations (P

Streptococcus pyogenes is a Gram-positive commensal bacterium that colonizes human tissue. When the host immune system is compromised or when the bacteria are able to penetrate the physical barrier of the skin, S. pyogenes can cause a wide range of diseases. Penetration of the skin barrier by GAS can cause bacteremia and severe invasive deep tissue infections, such as cellulitis, myositis, and necrotizing fasciitis. The most severe form of invasive infection results in clinical symptoms similar to staphylococcal toxic shock syndrome and was therefore described as streptococcal toxic shock syndrome (STSS). It is widely believed that the resurgence of invasive GAS infections is the result of an interplay between changes in bacterial virulence and host susceptibility. This chapter discusses a large number of secreted proteins (over 30) comprising the streptococcal superantigens and virulence factors including several adhesion molecules. GAS is associated with a number of severe invasive diseases, including cellulitis, myositis, and necrotizing fasciitis. Several lines of epidemiological, experimental, and clinical observations strongly imply that at least some SAgs play a pivotal role in the pathogenesis of STSS and possibly in other invasive GAS diseases.

Superantigens (SAgs) from group A streptococcus (GAS) are potent T cell mitogens, and have been suggested to play a role in severe streptococcal disease. Neutralizing antibodies protect against SAg-mediated disease and their levels should therefore be inversely related to severe streptococcal infection. Neutralizing anti-SAg titers in patients with severe GAS infection and patients without disease were compared in two separate groups. The first group comprised patients with invasive GAS disease from New Zealand European, Maori, and Pacific Island descent. The second group comprised Aboriginal Australian individuals with rheumatic heart disease and/or a past history of acute rheumatic fever. Patients sera were tested for their ability to neutralize T cell mitogenicity of recombinant streptococcal SAgs as a measure of functional SAg-neutralizing antibody concentration. In both studies, no inverse correlation was observed between disease and the level of serum SAg-neutralizing activity. Notably, much higher levels of natural immunity to all streptococcal SAgs were found in New Zealand Maori, New Zealand Pacific Island, and Aboriginal Australian individuals, suggesting a high degree of natural exposure and seroconversion in these groups compared to the New Zealand European cohort. Levels of serum antibodies against SAgs could not be used to predict disease susceptibility in groups with existing high levels of SAg-neutralizing antibodies.

Superantigens produced by Streptococcus pyogenes have been implicated with streptococcal toxic shock syndrome (STSS). We analyzed 19 acute-phase serum samples for mitogenic activity from patients with severe streptococcal disease. The serum samples from two patients in the acute phase of STSS showed strong proliferative activity. Streptococcal mitogenic exotoxin (SME) Z-1 and streptococcal pyrogenic exotoxin (SPE)-J were identified in one patient with peritonitis who recovered after 2 weeks in intensive care. SMEZ-16 was found in a second patient who died on the day of admission. Sequential serum samples taken on day 3 after admission from patient 1 showed clearance of mitogenic activity but absence of neutralizing anti-SMEZ antibodies. Serum samples taken on day 9 from this patient showed evidence of seroconversion with high levels of anti-SMEZ antibodies that neutralized SMEZ-1 and 12 other SMEZ-variants. These results imply that a high level of SMEZ production by group A streptococcus is a causative event in the onset and subsequent severity of STSS.

Two novel streptococcal superantigen genes (speLSeandspeMSe) were identified from theStreptococcus equigenome database at the Sanger Center. Genotyping of 8S. equiisolates and 40Streptococcus pyogenesisolates resulted in the detection of the orthologous genesspeLandspeMin a restricted number ofS. pyogenesisolates (15 and 5%, respectively). Surprisingly, the novel superantigen genes could not be found in any of the analyzedS. equiisolates. The results suggest that both genes are located on a mobile element that enables gene transfer between individual isolates and between streptococci from different Lancefield groups.S. equipyrogenic exotoxin L (SPE-LSe)/streptococcal pyrogenic exotoxin L (SPE-L) and SPE-MSe/SPE-M are most closely related to SMEZ, SPE-C, SPE-G, and SPE-J, but build a separate branch within this group. Recombinant SPE-L (rSPE-L) and rSPE-M were highly mitogenic for human peripheral blood lymphocytes, with half-maximum responses at 1 and 10 pg/ml, respectively. The results from competitive binding experiments suggest that both proteins bind major histocompatibility complex class II at the β-chain, but not at the α-chain. The most common targets for both toxins were human Vβ1.1 expressing T cells. Seroconversion against SPE-L and SPE-M was observed in healthy blood donors, suggesting that the toxins are expressed in vivo. Interestingly, thespeLgene is highly associated withS. pyogenesM89, a serotype that is linked to acute rheumatic fever in New Zealand.

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (KD, 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcαRI (CD89) and to a soluble form of the FcαRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (KD, 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.

The gene encoding streptococcal mitogenic exotoxin Z (SMEZ) was disrupted in Streptococcus pyogenes. Despite the presence of other superantigen genes, mitogenic responses in human and murine HLA-DQ transgenic cells were abrogated when cells were stimulated with supernatant from the smez− mutant compared with the parent strain. Remarkably, disruption of smez led to a complete inability to elicit cytokine production (TNF-α, lymphotoxin-α, IFN-γ, IL-1 and -8) from human cells, when cocultured with streptococcal supernatants. The potent effects of SMEZ were apparent even though transcription and expression of SMEZ were barely detectable. Human Vβ8+ T cell proliferation in response to S. pyogenes was SMEZ-dependent. Cells from HLA-DQ8 transgenic mice were 3 logs more sensitive to SMEZ-13 than cells from HLA-DR1 transgenic or wild-type mice. In the mouse, SMEZ targeted the human Vβ8+ TCR homologue, murine Vβ11, at the expense of other TCR T cell subsets. Expression of SMEZ did not affect bacterial clearance or survival from peritoneal streptococcal infection in HLA-DQ8 mice, though effects of SMEZ on pharyngeal infection are unknown. Infection did lead to a rise in Vβ11+ T cells in the spleen which was partly reversed by disruption of the smez gene. Most strikingly, a clear rise in murine Vβ4+ cells was seen in mice infected with the smez− mutant S. pyogenes strain, indicating a potential role for SMEZ as a repressor of cognate anti-streptococcal responses.

Neutrophils have been shown to express major histocompatibility complex class II (MHC II) after stimulation. However, reports concerning the functional effect of MCH II expression are still lacking. In our hands, granulocyte-monocyte colony-stimulating factor (GM-CSF) alone and in combination with interferon (IFN)-γ, but not IFN-γ or interleukin (IL)-3, induced a significant level of expression of human leukocyte antigen DR on neutrophils. The addition of staphylococcal enterotoxin E to neutrophils resulted in a significant increase in IL-8 production only after prestimulation with GM-CSF alone or in combination with IFN-γ but had no effect on neutrophils preincubated with IFN-γ alone or IL-3. Staphylococcal enterotoxin A, another bivalent superantigen, also stimulated production of IL-8 by preincubated polymorphonuclear neutrophils, whereas staphylococcal enterotoxin A mutants that are not able to cross-link MHC II molecules failed to induce IL-8 production. Taken together, our results clearly demonstrate that after induction of MHC II, neutrophils are able to respond to MHC II-specific stimulation. These findings support the ideas that the induced MHC II complex is completely functional and that neutrophils may be able to present antigens.