Staphylococcus aureus is a community- and hospital-associated pathogen of global significance. Despite several decades of investment there is no licenced vaccine and antimicrobial resistance is increasing, complicating effective treatment of infection. We have developed a novel fusion vaccine (PolySSL) containing mutated forms of three immune evasion factors, Staphylococcal Superantigen-Like (SSL) proteins 3, 7 and 11, that are common to all S. aureus isolates. Mice given the PolySSL vaccine have a robust specific serum IgG response, attenuated signs of infection, and a significantly reduced S. aureus tissue burden after an intra-peritoneal challenge with S. aureus. A comparison of six different versions of the vaccine (PolySSL 3711, 7113, 7311 and DualSSL 73, 311, 711) suggests inclusion of all three SSLs is required, the 7113 order produces the most significant reduction in bacterial liver and kidney burden, and that SSL7 is the most important component. The likely requirement for a multi-valent vaccine was examined by immunising groups of mice with six PolySSL7113 variants (adjuvanted with AdjuPhos) encompassing most S. aureus strains of clinical significance. SSL7 was confirmed to be the dominant antigen eliciting endpoint titres ≥105 irrespective of the variant used, whereas IgG responses to SSL3 and SSL11 were lower (∼103) and more variable. The highest inhibition of the interaction between the six wildtype variants of SSL7 and either IgA or complement C5 was typically obtained with variant-matched anti-sera. Conversely, anti-sera from these mice showed failed to stop SSL3 from blocking TLR2 signalling. An improved version of the PolySSL vaccine has been generated by altering the protein order, but these data suggest a multi-valent vaccine will be required to elicit robust inhibitory cross-variant antibody responses. A path to further improvement may be through use of a different adjuvant to broaden and enhance immunity to SSLs 3, 7 and 11.

Staphylococcus aureus is a significant bacterial pathogen in both community and hospital settings, and the escalation of antimicrobial-resistant strains is of immense global concern. Vaccination is an inviting long-term strategy to curb staphylococcal disease, but identification of an effective vaccine has proved to be challenging. Three well-characterized, ubiquitous, secreted immune evasion factors from the staphylococcal superantigen-like (SSL) protein family were selected for the development of a vaccine. Wild-type SSL3, 7 and 11, which inhibit signaling through Toll-like receptor 2, cleavage of complement component 5 and neutrophil function, respectively, were successfully combined into a stable, active fusion protein (PolySSL7311). Vaccination of mice with an attenuated form of the PolySSL7311 protein stimulated significantly elevated specific immunoglobulin G and splenocyte proliferation responses to each component relative to adjuvant-only controls. Vaccination with PolySSL7311, but not a mixture of the individual proteins, led to a > 10(2) reduction in S. aureus tissue burden compared with controls after peritoneal challenge. Comparable antibody responses were elicited after coadministration of the vaccine in either AddaVax (an analog of MF59) or an Alum-based adjuvant; but only AddaVax conferred a significant reduction in bacterial load, aligning with other studies that suggest both cellular and humoral immune responses are necessary for protective immunity to S. aureus. Anti-sera from mice immunized with PolySSL7311, but not individual proteins, partially neutralized the functional activities of SSL7. This study confirms the importance of these SSLs for the survival of S. aureus in vivo and suggests that PolySSL7311 is a promising vaccine candidate.

Staphylococcus aureus is a significant bacterial pathogen in both community and hospital settings, and the escalation of antimicrobial-resistant strains is of immense global concern. Vaccination is an inviting long-term strategy to curb staphylococcal disease, but identification of an effective vaccine has proved to be challenging. Three well-characterized, ubiquitous, secreted immune evasion factors from the staphylococcal superantigen-like (SSL) protein family were selected for the development of a vaccine. Wild-type SSL3, 7 and 11, which inhibit signaling through Toll-like receptor 2, cleavage of complement component 5 and neutrophil function, respectively, were successfully combined into a stable, active fusion protein (PolySSL7311). Vaccination of mice with an attenuated form of the PolySSL7311 protein stimulated significantly elevated specific immunoglobulin G and splenocyte proliferation responses to each component relative to adjuvant-only controls. Vaccination with PolySSL7311, but not a mixture of the individual proteins, led to a > 10 2 reduction in S. aureus tissue burden compared with controls after peritoneal challenge. Comparable antibody responses were elicited after coadministration of the vaccine in either AddaVax (an analog of MF59) or an Alum-based adjuvant; but only AddaVax conferred a significant reduction in bacterial load, aligning with other studies that suggest both cellular and humoral immune responses are necessary for protective immunity to S. aureus . Anti-sera from mice immunized with PolySSL7311, but not individual proteins, partially neutralized the functional activities of SSL7. This study confirms the importance of these SSLs for the survival of S. aureus in vivo and suggests that PolySSL7311 is a promising vaccine candidate.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

Objectives In preparation for the future arrival of a group A Streptococcus (GAS) vaccine, this study estimated the economic and health burdens of GAS diseases in New Zealand (NZ). Methods The annual incidence of GAS diseases was based on extrapolation of the average number of primary healthcare episodes managed each year in general practices (2014–2016) and on the average number of hospitalizations occurring each year (2005–2014). Disease incidence was multiplied by the average cost of diagnosing and managing an episode of disease at each level of care to estimate the annual economic burden. Results GAS affected 1.5% of the population each year, resulting in an economic burden of 29.2 million NZ dollars (2015 prices) and inflicting a health burden of 2373 disability-adjusted life years (DALYs). Children <5 years of age were the most likely age group to present for GAS-related healthcare. Presentations for superficial throat and skin infections (predominantly pharyngitis and impetigo) were more common than other GAS diseases. Cellulitis contributed the most to the total economic and health burdens. Invasive and immune-mediated diseases disproportionately contributed to the total economic and health burdens relative to their frequency of occurrence. Conclusion Preventing GAS diseases would have substantial economic and health benefits in NZ and globally.

Responses of solid tumors to chimeric antigen receptor (CAR) T cell therapy are often minimal. This is potentially due to a lack of sustained activation and proliferation of CAR T cells when encountering antigen in a profoundly immunosuppressive tumor microenvironment. In this study, we investigate if inducing an interaction between CAR T cells and antigen-presenting cells (APCs) in lymphoid tissue, away from an immunosuppressive microenvironment, could enhance solid-tumor responses. We combined CAR T cell transfer with the bacterial enterotoxin staphylococcal enterotoxin-B (SEB), which naturally links a proportion of T cell receptor (TCR) Vβ subtypes to MHC-II, present on APCs. CAR T cell proliferation and function was significantly enhanced by SEB. Solid tumor-growth inhibition in mice was increased when CAR T cells were administered in combination with SEB. CAR T cell expansion in lymphoid tissue was demonstrated, and inhibition of lymphocyte egress from lymph nodes using FTY720 abrogated the benefit of SEB. We also demonstrate that a bispecific antibody, targeting a c-Myc tag on CAR T cells and cluster of differentiation 40 (CD40), could also enhance CAR T cell activity and mediate increased antitumor activity of CAR T cells. These model systems serve as proof-of-principle that facilitating the interaction of CAR T cells with APCs can enhance their ability to mediate antitumor activity.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies.

Staphylococcus aureus and Group A Streptococcus (GAS) are common occupants of the tonsils and many strains produce potent exotoxins (mitogens) that directly target T cells, which could be a driver for tonsillar hyperplasia. Tonsil tissues from 41 patients were tested for these bacteria in conjunction with profiling of B and T cells by flow cytometry. S. aureus and GAS were detected in tonsil tissue from 44% and 7%, respectively, of patients by bacteriological culture; immuno-histology showed bacteria in close proximity to both B and T lymphocytes. The presence of tonsillar S. aureus did not alter B or T cell populations, whereas peripheral blood mucosal-associated invariant T (MAIT) cells were significantly increased in S. aureus culture positive individuals (p < 0.006). Alterations of tonsil CD4+ TCR Vβ family members relative to peripheral blood were evident in 29 patients. Three patients had strong TCR Vβ skewing indicative of recent exposure to superantigens, their tonsils contained mitogenic bacteria, and supernatants from these bacteria were used to partially recapitulate the skewing profile in vitro, supporting the notion that superantigens can target tonsillar T cells in situ. Tonsils are a reservoir for superantigen-producing bacteria with the capacity to alter the composition and function of key immune cells.

Previous studies have demonstrated that staphylococcal superantigen-like protein 7 (SSL7), a protein produced by the opportunistic bacterial pathogen Staphylococcus aureus, potently inhibits the formation of the complement membrane attack complex and release of C5a by binding to complement component 5 (C5). However, because of the predicted immunogenicity of SSL7 in humans, its potential as a new complement inhibitor for treating Complement-Mediated Diseases were uncertain. In this study, we found that administration of SSL7 efficiently protected red blood cells from complement-mediated lysis and reduced hemoglobinuria in a mouse model of paroxysmal nocturnal hemoglobinuria (PNH). Interestingly, at the dose required for effective complement inhibition in vivo, although repetitive administrations of SSL7 once every other week for six weeks elicited anti-SSL7 antibody production, SSL7 was still able to attenuate acute complement activation mediated Hemolysis and hemoglobinuria significantly in these mice. In addition, anterior chamber administration of SSL7 before repeatedly treating mice with SSL7 suppresses the production of SSL7-reactive antibodies and retain the treatment efficacy of SSL7. These results suggest that SSL7 could thus be developed as an economical alternative to the existing C5-targeted drug eculizumab, especially for controlling acute complement activation in catastrophic conditions such as drug-induced immune hemolytic anemia, ABO-incompatible red blood cell, transfusion, and cardiopulmonary bypass surgery.

BACKGROUND:Group A streptococcus (GAS) causes an exceptionally diverse range of diseases, raising questions about the optimal product characteristics of a commercially viable vaccine. The objectives of this study were to (1) estimate the current health and economic burdens caused by 24 diseases attributable to GAS each year in Australia and (2) use these estimates to explore the value of a GAS vaccine for different clinical indications, age schedules, and population groups. METHODS:For objective 1, we estimated the population heath and economic burdens by synthesising data from administrative databases, nationally representative surveys, literature reviews, public reimbursement schedules, and expert opinion. For objective 2, we modelled the prospective lifetime burden of GAS for all infants from birth, for children from 5 years of age, and for adults from 65 years of age. A vaccine was assumed to reduce each GAS disease by 70% for a period of 10 years, and the difference in outcomes between vaccinated and non-vaccinated cohorts were used to calculate the cost-effective value of vaccination. RESULTS:The annual health and economic burdens of GAS diseases totalled 23,528 disability-adjusted life years and AU$185.1 million in healthcare costs respectively; approximately half of each measure was due to cellulitis, followed by other skin infections and throat infections. Reducing the incidence of throat infections, skin infections, and cellulitis in non-Indigenous cohorts resulted in 30%, 33%, and 28% of the total vaccine value for an infant schedule (cost-effective vaccine price AU$260 per course); 47%, 26%, and 22% of the value for a child schedule (AU$289); and 2%, 15% and 74% for an adult schedule (AU$489). CONCLUSIONS:A vaccine that prevents GAS cellulitis and other skin infections, in addition to throat infections, would maximise its value and commercial viability, with a cost-effective price in line with other recently-licensed and funded vaccines in Australia.

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development.

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.

Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs) and the Staphylococcal Superantigen-Like (SSL) exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX) have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac)-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect S. aureus from destruction in a whole blood killing (WBK) assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the S. aureus exotoxin expression (Sae) two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX) reveals that SElX is a unique single-domain SAg. In summary, SElX is an 'SSL-like' SAg.

GroupA streptococci (GAS) cause a wide spectrum of diseases ranging from benign pharyngitis and skin infections to severe invasive disease and the immune sequelae rheumatic fever and rheumatic heart disease. Pharyngitis, one of the most frequent diseases caused by GAS, is highly prevalent in school-age children in temperate climates and a major cause of antibiotic use. An efficacious vaccine would reduce disease burden associated with pharyngitis and the need of care for sick children. Importantly, GAS pharyngitis is recognised as the main precursor for acute rheumatic fever so a vaccine that is efficacious against GAS pharyngitis should also prevent acute rheumatic fever and rheumatic heart disease. It may also prevent post-streptococcal glomerulonephritis and invasive disease since GAS pharyngitis is one of the precursors for these clinical syndromes. There has been no clearly articulated pathway for clinical trial design leading to GAS vaccine registration. This review outlines a clinical development strategy detailing the phases of development required for registration of a candidate GAS vaccine for GAS pharyngitis initially, followed by impetigo and associated sequelae. The major advantages of a strategy first focused on GAS pharyngitis is an early proof of principle, that can be followed by studies for other clinical syndromes. The end goal being the availability of a preventive tool for the most prevalent GAS-associated diseases globally.

Streptococcus pyogenes is an important global pathogen, causing considerable morbidity and mortality, especially in low and middle income countries where rheumatic heart disease and invasive infections are