Increased Breadth of Group A Streptococcus Antibody Responses in Children With Acute Rheumatic Fever Compared to Precursor Pharyngitis and Skin Infections.

BACKGROUND: Group A Streptococcus (GAS) causes superficial pharyngitis and skin infections as well as serious autoimmune sequelae such as acute rheumatic fever (ARF) and subsequent rheumatic heart disease. ARF pathogenesis remains poorly understood. Immune priming by repeated GAS infections is thought to trigger ARF, and there is growing evidence for the role of skin infections in this process. METHODS: We utilized our recently developed 8-plex immunoassay, comprising antigens used in clinical serology for diagnosis of ARF (SLO, DNase B, SpnA), and 5 conserved putative GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD, Group A carbohydrate), to characterize antibody responses in sera from New Zealand children with a range of clinically diagnosed GAS disease: ARF (n = 79), GAS-positive pharyngitis (n = 94), GAS-positive skin infection (n = 51), and matched healthy controls (n = 90). RESULTS: The magnitude and breadth of antibodies in ARF was very high, giving rise to a distinct serological profile. An average of 6.5 antigen-specific reactivities per individual was observed in ARF, compared to 4.2 in skin infections and 3.3 in pharyngitis. CONCLUSIONS: ARF patients have a unique serological profile, which may be the result of repeated precursor pharyngitis and skin infections that progressively boost antibody breadth and magnitude.

Combining antibody markers for serosurveillance of SARS-CoV-2 to estimate seroprevalence and time-since-infection.

Serosurveillance is an important epidemiologic tool for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), used to estimate infection rates and the degree of population immunity. There is no general agreement on which antibody biomarker(s) should be used, especially with the rollout of vaccines globally. Here, we used random forest models to demonstrate that a single spike or receptor-binding domain (RBD) antibody was adequate for classifying prior infection, while a combination of two antibody biomarkers performed better than any single marker for estimating time-since-infection. Nucleocapsid antibodies performed worse than spike or RBD antibodies for classification, but can be useful for estimating time-since-infection, and in distinguishing infection-induced from vaccine-induced responses. Our analysis has the potential to inform the design of serosurveys for SARS-CoV-2, including decisions regarding a number of antibody biomarkers measured.

Serological Profiling of Group A Streptococcus Infections in Acute Rheumatic Fever.

Rheumatic fever is a serious post-infectious sequela of group A Streptococcus (GAS). Prior GAS exposures were mapped in sera using a large panel of M-type specific peptides. Rheumatic fever patients had serological evidence of significantly more GAS exposures than matched controls suggesting immune priming by repeat infections contributes to pathogenesis.

Charting elimination in the pandemic: a SARS-CoV-2 serosurvey of blood donors in New Zealand.

New Zealand has a strategy of eliminating SARS-CoV-2 that has resulted in a low incidence of reported coronavirus-19 disease (COVID-19). The aim of this study was to describe the spread of SARS-CoV-2 in New Zealand via a nationwide serosurvey of blood donors. Samples (n = 9806) were collected over a month-long period (3 December 2020-6 January 2021) from donors aged 16-88 years. The sample population was geographically spread, covering 16 of 20 district health board regions. A series of Spike-based immunoassays were utilised, and the serological testing algorithm was optimised for specificity given New Zealand is a low prevalence setting. Eighteen samples were seropositive for SARS-CoV-2 antibodies, six of which were retrospectively matched to previously confirmed COVID-19 cases. A further four were from donors that travelled to settings with a high risk of SARS-CoV-2 exposure, suggesting likely infection outside New Zealand. The remaining eight seropositive samples were from seven different district health regions for a true seroprevalence estimate, adjusted for test sensitivity and specificity, of 0.103% (95% confidence interval, 0.09-0.12%). The very low seroprevalence is consistent with limited undetected community transmission and provides robust, serological evidence to support New Zealand's successful elimination strategy for COVID-19.

The Limitations of the Rheumatogenic Concept for Group A Streptococcus: Systematic Review and Genetic Analysis.

BACKGROUND: The concept that a minority of group A streptococcus (GAS) emm types are more "rheumatogenic" than others has been widely disseminated. We aimed to provide a comprehensive list of acute rheumatic fever-associated GAS isolates and assess the presence of associated rheumatogenic motifs. METHODS: Articles reporting GAS emm-type or emm-type-specific antibody responses associated with rheumatic fever were identified from 1 January 1944 to 31 July 2018. The revised Jones criteria were used to define rheumatic fever with a maximum period of 4 weeks between disease onset and microbiological characterization. A database of 175 representative M-protein sequences was used to analyze the protein diversity of rheumatic fever-associated strains in a phylogenetic tree and to identify the presence of 10 previously recognized rheumatogenic motifs. RESULTS: We included 411 cases of rheumatic fever, for which microbiological characterization identified 73 different emm types associated with the disease. The classic rheumatogenic emm types represented only 12.3% of the 73 emm types and were responsible for 31.6% of the 411 clinical cases. Rheumatic fever-associated emm types were disseminated throughout the phylogeny, suggesting they belong to various genetic backgrounds. Rheumatic fever-associated motifs were present in only 15.1% of the rheumatic fever-associated emm types and only 24.8% of clinical cases. CONCLUSIONS: The concept of rheumatogenicity should be extended to include strains other than those classically described. Our results highlight significant knowledge gaps in the understanding of rheumatic fever pathogenesis and suggest that a GAS vaccine candidate should offer broad coverage against a variety of GAS genetic variants in order to protect against this serious sequela.

Systems immunology reveals a linked IgG3-C4 response in patients with acute rheumatic fever.

Acute rheumatic fever (ARF) and chronic rheumatic heart disease (RHD) are autoimmune sequelae of a Group A streptococcal infection with significant global mortality and poorly understood pathogenesis. Immunoglobulin and complement deposition were observed in ARF/RHD valve tissue over 50 years ago, yet contemporary investigations have been lacking. This study applied systems immunology to investigate the relationships between the complement system and immunoglobulin in ARF. Patients were stratified by C-reactive protein (CRP) concentration into high (≥10 µg mL-1 ) and low (<10 µg mL-1 ) groups to distinguish those with clinically significant inflammatory processes from those with abating inflammation. The circulating concentrations of 17 complement factors and six immunoglobulin isotypes and subclasses were measured in ARF patients and highly matched healthy controls using multiplex bead-based immunoassays. An integrative statistical approach combining feature selection and principal component analysis revealed a linked IgG3-C4 response in ARF patients with high CRP that was absent in controls. Strikingly, both IgG3 and C4 were elevated above clinical reference ranges, suggesting these features are a marker of ARF-associated inflammation. Humoral immunity in response to M protein, an antigen implicated in ARF pathogenesis, was completely polarized to IgG3 in the patient group. Furthermore, the anti-M-protein IgG3 response was correlated with circulating IgG3 concentration, highlighting a potential role for this potent immunoglobulin subclass in disease. In conclusion, a linked IgG3-C4 response appears important in the initial, inflammatory stage of ARF and may have immediate utility as a clinical biomarker given the lack of specific diagnostic tests currently available.

Development and Evaluation of a New Triplex Immunoassay That Detects Group A Streptococcus Antibodies for the Diagnosis of Rheumatic Fever.

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 <28 and 28+ day groups. However, for anti-SpnA, there was a significant decrease (P < 0.05) in the 28+ day group, indicative of faster anti-SpnA antibody decay. Anti-SpnA immunokinetics support very recent group A Streptococcus infection and may assist in diagnostic classification of ARF. Further, bead-based assays enable streptococcal serology to be performed efficiently in a high-throughput manner.

Group A Streptococcus T Antigens Have a Highly Conserved Structure Concealed under a Heterogeneous Surface That Has Implications for Vaccine Design.

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.

Understanding group A streptococcal pharyngitis and skin infections as causes of rheumatic fever: protocol for a prospective disease incidence study.

BACKGROUND: Group A Streptococcal (GAS) infections cause the autoimmune disease acute rheumatic fever (ARF), which can progress to chronic rheumatic heart disease (RHD). Treating pharyngitis caused by GAS with antibiotics is important in preventing ARF. However, it is difficult to distinguish these infections from GAS carriers. There is growing evidence for GAS skin infections as a cause of ARF. This study will identify the incidence of true GAS pharyngitis and serological responses to GAS skin infections. The effectiveness of antibiotics for these conditions will be explored, and modifiable risk factors. Serum antibody titres indicating the upper limits of normal (ULN for ASO/ADB antibodies) will be established alongside carriage rates in asymptomatic children. METHODS: This is a prospective disease incidence study, with an associated case-control study. The study population includes 1000 children (5-14 years) from Auckland, New Zealand, 800 of whom have visited their healthcare professional, resulting in a throat or skin swab for GAS, and 200 who are asymptomatic. The conditions of interest are GAS throat swab positive pharyngitis (n = 200); GAS carriage (n = 200); GAS negative throat swab (n = 200); GAS skin infections (n = 200); and asymptomatic controls (n = 200). All participants, except asymptomatic controls, will have acute and convalescent serological testing for ASO/ADB titres (collected < 9 days, and 2-4 weeks following symptom onset, respectively), alongside viral PCR from throat swabs. Asymptomatic controls will have ASO/ADB titres measured in one blood specimen and a throat swab for microbial culture. Caregivers of children will be interviewed using a questionnaire and any GAS isolates identified will be emm typed. The persistence of GAS antibodies will also be investigated. DISCUSSION: Findings from this study will fill critical gaps in scientific knowledge to better understand the pathophysiology of ARF, improve clinical management of GAS infections, and design more effective ARF prevention programmes. In particular it will measure the incidence of true, serologically confirmed GAS pharyngitis; assess the immune response to GAS skin infections and its role as a cause of ARF; examine the effectiveness of oral antibiotics for treating GAS pharyngitis and carriage; and identify whether risk factors for GAS infections might provide intervention points for reducing ARF.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

Comparative M-protein analysis of Streptococcus pyogenes from pharyngitis and skin infections in New Zealand: Implications for vaccine development.

BACKGROUND: Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are responsible for a significant disease burden amongst Maori and Pacific populations in New Zealand (NZ). However, contemporary data are lacking regarding circulating group A Streptococcal (GAS) strains in NZ. Such information is important in guiding vaccine development. METHODS: GAS isolates from April to June 2015 were recovered from skin and pharyngeal samples from children living in areas of high social deprivation in Auckland, NZ, a significant proportion of which are Maori or Pacific. These children are among the highest risk group for developing ARF. Isolates were compared to concurrently collected pharyngeal isolates from Dunedin, NZ, where both the proportion of Maori and Pacific children and risk of developing ARF is low. Emm typing, emm cluster typing and theoretical coverage of the 30-valent vaccine candidate were undertaken as previously described. RESULTS: A high diversity of emm types and a high proportion of emm-pattern D and cluster D4 isolates were detected amongst both skin and pharyngeal isolates in children at high risk of ARF. Pharyngeal isolates from children at low risk of ARF within the same country were significantly less diverse, less likely to be emm pattern D, and more likely to be theoretically covered by the 30-valent M protein vaccine. CONCLUSIONS: The high proportion of emm pattern D GAS strains amongst skin and pharyngeal isolates from children at high risk of ARF raises further questions about the role of skin infection in ARF pathogenesis. Emm types and emm clusters differed considerably between ARF endemic and non-endemic settings, even within the same country. This difference should be taken into account for vaccine development.

M-Protein Analysis of Streptococcus pyogenes Isolates Associated with Acute Rheumatic Fever in New Zealand.

We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.

Crystal structure of PhnF, a GntR-family transcriptional regulator of phosphate transport in Mycobacterium smegmatis.

Bacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

Structural conservation, variability, and immunogenicity of the T6 backbone pilin of serotype M6 Streptococcus pyogenes.

Group A streptococcus (GAS; Streptococcus pyogenes) is a Gram-positive human pathogen that causes a broad range of diseases ranging from acute pharyngitis to the poststreptococcal sequelae of acute rheumatic fever. GAS pili are highly diverse, long protein polymers that extend from the cell surface. They have multiple roles in infection and are promising candidates for vaccine development. This study describes the structure of the T6 backbone pilin (BP; Lancefield T-antigen) from the important M6 serotype. The structure reveals a modular arrangement of three tandem immunoglobulin-like domains, two with internal isopeptide bonds. The T6 pilin lysine, essential for polymerization, is located in a novel VAKS motif that is structurally homologous to the canonical YPKN pilin lysine in other three- and four-domain Gram-positive pilins. The T6 structure also highlights a conserved pilin core whose surface is decorated with highly variable loops and extensions. Comparison to other Gram-positive BPs shows that many of the largest variable extensions are found in conserved locations. Studies with sera from patients diagnosed with GAS-associated acute rheumatic fever showed that each of the three T6 domains, and the largest of the variable extensions (V8), are targeted by IgG during infection in vivo. Although the GAS BP show large variations in size and sequence, the modular nature of the pilus proteins revealed by the T6 structure may aid the future design of a pilus-based vaccine.

Reduction in Acute Post-Streptococcal Glomerulonephritis Incidence in Counties Manukau, New Zealand, after the COVID-19 Pandemic.

The COVID-19 pandemic has altered the epidemiology of many common childhood infections, including Group A streptococcal (GAS) disease. Acute post-streptococcal glomerulonephritis (APSGN) is a nonsuppurative complication of GAS pharyngitis and pyoderma. It remains the most common cause of pediatric acute glomerulonephritis globally. In Counties Manukau, New Zealand, APSGN rates have previously been shown to be the highest in the country, with marked ethnic and socioeconomic disparities. We performed a retrospective review of children aged 0-14 years who were discharged from Kidz First Hospital, Counties Manukau, between 2015 and 2023 and met the Strep A Vaccine Global Consortium consensus definition of APSGN. We describe a marked, sustained reduction in APSGN hospitalizations, temporally associated with the COVID-19 pandemic. This ongoing reduction in APSGN incidence is notable in light of contrasting reports of increasing incidence of rheumatic fever in New Zealand and invasive GAS disease internationally.

Development and characterization of a hemolysis inhibition assay to determine functionality of anti-Streptolysin O antibodies in human sera.

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies.

Human antibody profiling technologies for autoimmune disease.

Autoimmune diseases are caused by the break-down in self-tolerance mechanisms and can result in the generation of autoantibodies specific to human antigens. Human autoantigen profiling technologies such as solid surface arrays and display technologies are powerful high-throughput technologies utilised to discover and map novel autoantigens associated with disease. This review compares human autoantigen profiling technologies including the application of these approaches in chronic and post-infectious autoimmune disease. Each technology has advantages and limitations that should be considered when designing new projects to profile autoantibodies. Recent studies that have utilised these technologies across a range of diseases have highlighted marked heterogeneity in autoantibody specificity between individuals as a frequent feature. This individual heterogeneity suggests that epitope spreading maybe an important mechanism in the pathogenesis of autoimmune disease in general and likely contributes to inflammatory tissue damage and symptoms. Studies focused on identifying autoantibody biomarkers for diagnosis should use targeted data analysis to identify the rarer public epitopes and antigens, common between individuals. Thus, utilisation of human autoantigen profiling technology, combined with different analysis approaches, can illuminate both pathogenesis and biomarker discovery.