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Streptococcus. pyogenes (Strep A) infections result in a vastly underestimated burden of acute and chronic disease globally. The Strep A Vaccine Global Consortium (SAVAC) mission is to accelerate the development of safe, effective and affordable S. pyogenes vaccines. The safety of vaccine recipients is of paramount importance. A single S. pyogenes vaccine clinical trial conducted in the 1960s raised important safety concerns. A SAVAC Safety Working Group was established to review the safety assessment methodology and results of more recent early phase clinical trials and to consider future challenges for vaccine safety assessments across all phases of vaccine development. No clinical or biological safety signals were detected in any of these early phase trials in the modern era. Improvements in vaccine safety assessments need further consideration, particularly for pediatric clinical trials, large-scale efficacy trials, and preparation for post-marketing pharmacovigilance.

Background: Streptococcus pyogenes is a leading cause of infection-related morbidity and mortality. A reinvigorated vaccine development effort calls for new clinically relevant human S pyogenes experimental infection models to support proof of concept evaluation of candidate vaccines. We describe the initial Controlled Human Infection for Vaccination Against S pyogenes (CHIVAS-M75) study, in which we aimed to identify a dose of emm75 S pyogenes that causes acute pharyngitis in at least 60% of volunteers when applied to the pharynx by swab. Methods: This observational, dose-finding study was done in a clinical trials facility in Melbourne (VIC, Australia). Groups of healthy volunteers aged 18–40 years, at low risk of complicated S pyogenes disease, and without high type-specific anti-emm75 IgG antibodies against the challenge strain were challenged and closely monitored as inpatients for up to 6 days, and then as outpatients for 6 months. Antibiotics were started upon diagnosis (clinical signs and symptoms of pharyngitis and a positive rapid molecular test) or after 5 days in those without pharyngitis. Rapid test results were confirmed by standard bacterial culture. After a sentinel participant, cohorts of five and then ten participants were challenged, with protocol-directed dose-escalation or de-escalation for subsequent cohorts. The primary outcome was the proportion of participants at each dose level with pharyngitis by day 5 after challenge. The study is registered with ClinicalTrials.gov, NCT03361163. Findings: Between July 10, 2018, and Sept 23, 2019, 25 healthy adults were challenged with emm75 S pyogenes and included in analyses. Pharyngitis was diagnosed in 17 (85%; 95% CI 62–97) of 20 participants at the starting dose level (1–3 × 105 colony-forming units [CFU]/mL). This high proportion prompted dose de-escalation. At the lower dose level (1–3 × 104 CFU/mL), pharyngitis was diagnosed in one of five participants. Immunological, biochemical, and microbiological results supported the clinical picture, with acute symptomatic pharyngitis characterised by pharyngeal colonisation by S pyogenes accompanied by significantly elevated C-reactive protein and inflammatory cytokines (eg, interferon-γ and interleukin-6), and modest serological responses to streptolysin O and deoxyribonuclease B. There were no severe (grade 3) or serious adverse events related to challenge. Interpretation: We have established a reliable pharyngitis human infection model with reassuring early safety findings to accelerate development of vaccines and other interventions to control disease due to S pyogenes. Funding: Australian National Health and Medical Research Council., SCOPUS: ar.j, info:eu-repo/semantics/published

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). To examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ≥60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1–50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.

Objectives: Group A β-hemolytic Streptococcus (GAS), also known as Streptococcus pyogenes, is responsible for an annual 600 million cases of acute pharyngitis globally, with 92% of those infections occurring in low-resource settings. Further knowledge of the acute streptococcal pharyngitis burden in low-resource settings is essential if serious post-streptococcal complications – rheumatic fever (RF) and its long-term sequel rheumatic heart disease (RHD) – are to be prevented. Methods: Two studies were conducted in school-aged children (5–16 years): a cross-sectional study of streptococcal pharyngeal carriage followed by a prospective cohort study of streptococcal sore throat over 4 weeks from March to April 2017. Results: The cross-sectional study revealed an overall prevalence of GAS carriage of 15.9% (79/496, 95% confidence interval 12.8–19.5%). Among 532 children enrolled in the prospective cohort study, 358 (67%) reported 528 sore throats, with 221 (41.1%) experiencing at least one GAS-positive sore throat. The overall GAS-positive rate for sore throat was 41.8% (221/528). Conclusions: The GAS pharyngeal carriage rates seen in Uganda (15.9%, 95% confidence interval 12.8–19.5%) are higher than the most recent pooled results globally, at 12% (range 6–28%). Additionally, pilot data suggest a substantially higher percentage of sore throat that was GAS-positive (41.8%) compared to pooled global rates when active recruitment is employed. Keywords: Group A β-hemolytic Streptococcus, Uganda, Rheumatic fever, Rheumatic heart disease

Group A streptococcus (Strep A) surface M protein, an α-helical coiled-coil dimer, is a vaccine target and a major determinant of streptococcal virulence. The sequence-variable N-terminal region of the M protein defines the M type and also contains epitopes that promote opsonophagocytic killing of streptococci. Recent reports have reported considerable cross-reactivity among different M types, suggesting the prospect of identifying cross-protective epitopes that would constitute a broadly protective multivalent vaccine against Strep A isolates. Here, we have used a combination of immunological assays, structural biology, and cheminformatics to construct a recombinant M protein-based vaccine that included six Strep A M peptides that were predicted to elicit antisera that would cross-react with an additional 15 nonvaccine M types of Strep A. Rabbit antisera against this recombinant vaccine cross-reacted with 10 of the 15 nonvaccine M peptides. Two of the five nonvaccine M peptides that did not cross-react shared high sequence identity (≥50%) with the vaccine peptides, implying that high sequence identity alone was insufficient for cross-reactivity among the M peptides. Additional structural analyses revealed that the sequence identity at corresponding polar helical-wheel heptad sites between vaccine and nonvaccine peptides accurately distinguishes cross-reactive from non-cross-reactive peptides. On the basis of these observations, we developed a scoring algorithm based on the sequence identity at polar heptad sites. When applied to all epidemiologically important M types, this algorithm should enable the selection of a minimal number of M peptide-based vaccine candidates that elicit broadly protective immunity against Strep A.

Background Streptococcus pyogenes (group A Streptococcus, Strep A) is a widespread pathogen that continues to pose a significant threat to human health. The development of a Strep A vaccine remains an unmet global health need. One of the major vaccine strategies is the use of M protein, which is a primary virulence determinant and protective antigen. Multivalent recombinant M protein vaccines are being developed with N-terminal M peptides that contain opsonic epitopes but do not contain human tissue cross-reactive epitopes. Methods We completed a Phase I trial of a recombinant 30-valent M protein-based Strep A vaccine (Strep A vaccine, StreptAnova™) comprised of four recombinant proteins containing N-terminal peptides from 30 M proteins of common pharyngitis and invasive and/or rheumatogenic serotypes, adjuvanted with aluminum hydroxide. The trial was observer-blinded and randomized in a 2:1 ratio for intramuscular administration of Strep A vaccine or an alum-based comparator in healthy adult volunteers, at 0, 30 and 180 days. Primary outcome measures were assessments of safety, including assays for antibodies that cross-reacted with host tissues, and immunogenicity assessed by ELISA with the individual vaccine peptides and by opsonophagocytic killing (OPK) assays in human blood. Results Twenty-three Strep A-vaccinated participants and 13 controls completed the study. The Strep A vaccine was well-tolerated and there was no clinical evidence of autoimmunity and no laboratory evidence of tissue cross-reactive antibodies. The vaccine was immunogenic and elicited significant increases in geometric mean antibody levels to 24 of the 30 component M antigens by ELISA. Vaccine-induced OPK activity was observed against selected M types of Strep A in vaccinated participants that seroconverted to specific M peptides. Conclusion The Strep A vaccine was well tolerated and immunogenic in healthy adults, providing strong support for further clinical development. [ClinicalTrials.gov NCT02564237].

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. One obstacle to the development of M protein–based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.

Rheumatic heart disease (RHD) affects ≈40 million people and claims nearly 300 000 lives each year. The historic passing of a World Health Assembly resolution on RHD in 2018 now mandates a coordinated global response. The American Heart Association is committed to serving as a global champion and leader in RHD care and prevention. Here, we pledge support in 5 key areas: (1) professional healthcare worker education and training, (2) technical support for the implementation of evidence-based strategies for rheumatic fever/RHD prevention, (3) access to essential medications and technologies, (4) research, and (5) advocacy to increase global awareness, resources, and capacity for RHD control. In bolstering the efforts of the American Heart Association to combat RHD, we hope to inspire others to collaborate, communicate, and contribute.

BackgroundAnemm-cluster based system was proposed as a standard typing scheme to facilitate and enhance future studies of Group A Streptococcus (Strep A) epidemiological surveillance, M protein function and vaccine development strategies. We provide an evidence-based distribution of Strep Aemmclusters in Africa and assess the potential coverage of the new 30-valent vaccine in terms of an emm cluster-based approach.MethodTwo reviewers independently assessed studies retrieved from a comprehensive search and extracted relevant data. Meta-analyses were performed (random effects model) to aggregateemmcluster prevalence estimates.ResultsEight studies (n=1,595 isolates) revealed the predominantemmclusters as E6 (18%, 95% confidence interval (CI), 12.6; 24.0%), followed by E3 (14%, 95%CI, 11.2; 17.4%) and E4 (13%, 95%CI, 9.5; 16.0%). There is negligible variation inemmclusters as regards regions, age and socio-economic status across the continent. Considering anemmcluster-based vaccine strategy, which assumes cross-protection within clusters, the 30-valent vaccine currently in clinical development, would provide hypothetical coverage to 80.3% of isolates in Africa.ConclusionThis systematic review indicates the most predominant Strep Aemmcluster in Africa is E6 followed by E3, E4 and D4. The current 30-valent vaccine would provide considerable coverage across the diversity ofemmcluster types in Africa. Future efforts could be directed toward estimating the overall potential coverage of the new 30-valent vaccine based on cross-opsonization studies with representative panels of Strep A isolates from populations at highest risk for Strep A diseases.ImportanceLow vaccine coverage is of grave public health concern, particularly in developing countries where epidemiological data are often absent. To inform vaccine development for group A streptococcus (Strep A), we report on the epidemiology of the M Protein emm clusters from Strep A infections in Africa, where Strep A-related illnesses and their sequalae including rheumatic fever and rheumatic heart disease, are of a high burden. This first report of emm clusters across the continent indicate a high probably of coverage by the M Protein-based vaccine currently undergoing testing, were an emm-cluster based approach to be used.

Here we adapt and evaluate a full-face snorkel mask for use as personal protective equipment (PPE) for health care workers, who lack appropriate alternatives during the COVID-19 crisis in the spring of 2020. The design (referred to as Pneumask) consists of a custom snorkel-specific adapter that couples the snorkel-port of the mask to a rated filter (either a medical-grade ventilator inline filter or an industrial filter). This design has been tested for the sealing capability of the mask, filter performance, CO2 buildup and clinical usability. These tests found the Pneumask capable of forming a seal that exceeds the standards required for half-face respirators or N95 respirators. Filter testing indicates a range of options with varying performance depending on the quality of filter selected, but with typical filter performance exceeding or comparable to the N95 standard. CO2 buildup was found to be roughly equivalent to levels found in half-face elastomeric respirators in literature. Clinical usability tests indicate sufficient visibility and, while speaking is somewhat muffled, this can be addressed via amplification (Bluetooth voice relay to cell phone speakers through an app) in noisy environments. We present guidance on the assembly, usage (donning and doffing) and decontamination protocols. The benefit of the Pneumask as PPE is that it is reusable for longer periods than typical disposable N95 respirators, as the snorkel mask can withstand rigorous decontamination protocols (that are standard to regular elastomeric respirators). With the dire worldwide shortage of PPE for medical personnel, our conclusions on the performance and efficacy of Pneumask as an N95-alternative technology are cautiously optimistic.Key pointsFull-face snorkel masks are adapted for use as Personal Protective Equipment during the COVID-19 crisis, using a custom adapter that facilitates the attachment of inline medical-grade respiratory filters or NIOSH industrial respirator filters. This solution was designed as a reusable stopgap solution for healthcare workers to help address the short-term global N95 respirator shortage.

PURPOSE OF REVIEW: There is a global need for well tolerated, effective, and affordable vaccines to prevent group A streptococcal infections and their most serious complications. The aim of this review is to highlight the recent progress in the identification of promising vaccine antigens and new approaches to vaccine design that address the complexities of group A streptococcal pathogenesis and epidemiology. RECENT FINDINGS: Combination vaccines containing multiple shared, cross-protective antigens have proven efficacious in mouse and nonhuman primate models of infection. The development of complex multivalent M protein-based vaccines is continuing and several have progressed through early-stage human clinical trials. Formulations of vaccines containing universal T-cell epitopes, toll-like receptor agonists, and other adjuvants more potent than alum have been shown to enhance protective immunogenicity. Although the group A streptococcal vaccine antigen landscape is populated with a number of potential candidates, the clinical development of vaccines has been impeded by a number of factors. There are now concerted global efforts to raise awareness about the need for group A streptococcal vaccines and to support progress toward eventual commercialization and licensure. SUMMARY: Preclinical antigen discovery, vaccine formulation, and efficacy studies in animal models have progressed significantly in recent years. There is now a need to move promising candidates through the clinical development pathway to establish their efficacy in preventing group A streptococcal infections and their complications.

Protein subunit vaccines are often preferred because of their protective efficacy and safety. Lactic acid bacteria expressing heterologous antigens constitute a promising approach to vaccine development. However, their safety in terms of toxicity and bacterial clearance must be evaluated. Anti-Streptococcus pyogenes (S. pyogenes) vaccines face additional safety concerns because they may elicit autoimmune responses. The assessment of toxicity, clearance and autoimmunity of an anti-streptococcal vaccine based on Lactococcus lactis (L. lactis) expressing 10 different M protein fragments from S. pyogenes (L. lactis-Mx10) is here reported. Clearance of L. lactis from the oropharynges of immunocompetent mice and mice devoid of T/B lymphocytes mice was achieved without using antibiotics. The absence of autoimmune responses against human tissues was demonstrated with human brain, heart and kidney. Assessment of toxicity showed that leucocyte counts and selected serum biochemical factors were not affected in L. lactis-Mx10-immunized mice. In contrast, mice immunized with L. lactis wild type vector (L. lactis-WT) showed increased neutrophil and monocyte counts and altered histopathology of lymph nodes, lungs and nasal epithelium. Two days after immunization, L. lactis-Mx10-immunized and L. lactis-WT-immunized mice weighed significantly less than unimmunized mice. However, both groups of immunized mice recovered their body weights by Day 6. Our results demonstrate that L. lactis-WT, but not the vaccine L. lactis-Mx10, induces alterations in certain hematologic and histopathological variables. We consider these data a major contribution to data on L. lactis as a bacterial vector for vaccine delivery.

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N-terminal fragment of M protein, has been developed. Live bacterial-vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10-valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three- to 10-fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10-immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10-immunized mice did not differ significantly from that of unimmunized mice. Mx-10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

Existing clinical decision rules (CDRs) to diagnose group A streptococcal (GAS) pharyngitis have not been validated in sub-Saharan Africa. We developed a locally applicable CDR while evaluating existing CDRs for diagnosing GAS pharyngitis in South African children. We conducted a prospective cohort study and enrolled 997 children 3–15 years of age presenting to primary care clinics with a complaint of sore throat, and whose parents provided consent. Main outcome measures were signs and symptoms of pharyngitis and a positive GAS culture from a throat swab. Bivariate and multivariate analyses were used to develop the CDR. In addition, the diagnostic effectiveness of 6 existing rules for predicting a positive culture in our cohort was assessed. A total of 206 of 982 children (21%) had a positive GAS culture. Tonsillar swelling, tonsillar exudates, tender or enlarged anterior cervical lymph nodes, absence of cough and absence of rhinorrhea were associated with positive cultures in bivariate and multivariate analyses. Four variables (tonsillar swelling and one of tonsillar exudate, no rhinorrhea, no cough), when used in a cumulative score, showed 83.7% sensitivity and 32.2% specificity for GAS pharyngitis. Of existing rules tested, the rule by McIsaac et al had the highest positive predictive value (28%), but missed 49% of the culture-positive children who should have been treated. The new 4-variable CDR for GAS pharyngitis (ie, tonsillar swelling and one of tonsillar exudate, no rhinorrhea, no cough) outperformed existing rules for GAS pharyngitis diagnosis in children with symptomatic sore throat in Cape Town.

The development of a vaccine for group A streptococcus (GAS) is of paramount importance given that GAS infections cause more than 500,000 deaths annually across the world. This prospective passive surveillance laboratory study evaluated the potential coverage of the M protein-based vaccine currently under development. While a number of GAS strains isolated from this sub-Sahara African study were included in the current vaccine formulation, we nevertheless report that potential vaccine coverage for GAS infection in our setting was approximately 60%, with four of the most prevalent strains not included. This research emphasizes the need to reformulate the vaccine to improve coverage in areas where the burden of disease is high., Group A streptococcus (GAS) is responsible for a wide range of noninvasive group A streptococcal (non-iGAS) and invasive group A streptococcal (iGAS) infections. Information about the emm type variants of the M protein causing GAS disease is important to assess potential vaccine coverage of a 30-valent vaccine under development, particularly with respect to how they compare and contrast with non-iGAS isolates, especially in regions with a high burden of GAS. We conducted a prospective passive surveillance study of samples from patients attending public health facilities in Cape Town, South Africa. We documented demographic data and clinical presentation. emm typing was conducted using CDC protocols. GAS was commonly isolated from pus swabs, blood, deep tissue, and aspirates. Clinical presentations included wound infections (20%), bacteremia (15%), abscesses (9%), and septic arthritis (8%). Forty-six different emm types were identified, including M76 (16%), M81 (10%), M80 (6%), M43 (6%), and M183 (6%), and the emm types were almost evenly distributed between non-iGAS and iGAS isolates. There was a statistically significant association with M80 in patients presenting with noninvasive abscesses. Compared to the 30-valent vaccine under development, the levels of potential vaccine coverage for non-iGAS and iGAS infection were 60% and 58%, respectively, notably lower than the coverage in developed countries; five of the most prevalent emm types, M76, M81, M80, M43, and M183, were not included. The emm types from GAS isolated from patients with invasive disease did not differ significantly from those from noninvasive disease cases. There is low coverage of the multivalent M protein vaccine in our setting, emphasizing the need to reformulate the vaccine to improve coverage in areas where the burden of disease is high. IMPORTANCE The development of a vaccine for group A streptococcus (GAS) is of paramount importance given that GAS infections cause more than 500,000 deaths annually across the world. This prospective passive surveillance laboratory study evaluated the potential coverage of the M protein-based vaccine currently under development. While a number of GAS strains isolated from this sub-Sahara African study were included in the current vaccine formulation, we nevertheless report that potential vaccine coverage for GAS infection in our setting was approximately 60%, with four of the most prevalent strains not included. This research emphasizes the need to reformulate the vaccine to improve coverage in areas where the burden of disease is high.

Group A streptococcus (GAS) is responsible for a wide range of noninvasive group A streptococcal (non-iGAS) and invasive group A streptococcal (iGAS) infections. Information about the emm type variants of the M protein causing GAS disease is important to assess potential vaccine coverage of a 30-valent vaccine under development, particularly with respect to how they compare and contrast with non-iGAS isolates, especially in regions with a high burden of GAS. We conducted a prospective passive surveillance study of samples from patients attending public health facilities in Cape Town, South Africa. We documented demographic data and clinical presentation. emm typing was conducted using CDC protocols. GAS was commonly isolated from pus swabs, blood, deep tissue, and aspirates. Clinical presentations included wound infections (20%), bacteremia (15%), abscesses (9%), and septic arthritis (8%). Forty-six different emm types were identified, including M76 (16%), M81 (10%), M80 (6%), M43 (6%), and M183 (6%), and the emm types were almost evenly distributed between non-iGAS and iGAS isolates. There was a statistically significant association with M80 in patients presenting with noninvasive abscesses. Compared to the 30-valent vaccine under development, the levels of potential vaccine coverage for non-iGAS and iGAS infection were 60% and 58%, respectively, notably lower than the coverage in developed countries; five of the most prevalent emm types, M76, M81, M80, M43, and M183, were not included. The emm types from GAS isolated from patients with invasive disease did not differ significantly from those from noninvasive disease cases. There is low coverage of the multivalent M protein vaccine in our setting, emphasizing the need to reformulate the vaccine to improve coverage in areas where the burden of disease is high. IMPORTANCE The development of a vaccine for group A streptococcus (GAS) is of paramount importance given that GAS infections cause more than 500,000 deaths annually across the world. This prospective passive surveillance laboratory study evaluated the potential coverage of the M protein-based vaccine currently under development. While a number of GAS strains isolated from this sub-Sahara African study were included in the current vaccine formulation, we nevertheless report that potential vaccine coverage for GAS infection in our setting was approximately 60%, with four of the most prevalent strains not included. This research emphasizes the need to reformulate the vaccine to improve coverage in areas where the burden of disease is high.

Group A Streptococcus (GAS) is a highly-adapted and human-restricted pathogen responsible for a high global burden of disease across a diverse clinical spectrum. Vaccine development has been impeded by scientific, regulatory, and commercial obstacles. Human infection studies (HIS) are increasingly contributing to drug, diagnostics, and vaccine development, reducing uncertainty at early stages, especially for pathogens with animal models that incompletely reproduce key elements of human disease. We review the small number of historical GAS HIS and present the study protocol for a dose-ranging inpatient study in healthy adults. The primary objective of the study is to establish a new GAS pharyngitis HIS with an attack rate of at least 60% as a safe and reliable platform for vaccine evaluation and pathogenesis research. According to an adaptive dose-ranging study design, emm75 GAS doses manufactured in keeping with principles of Good Manufacturing Practice will be directly applied by swab to the pharynx of carefully screened healthy adult volunteers at low risk of severe complicated GAS disease. Participants will remain as closely monitored inpatients for up to six days, observed for development of the primary outcome of acute symptomatic pharyngitis, as defined by clinical and microbiological criteria. All participants will be treated with antibiotics and followed as outpatients for six months. An intensive sampling schedule will facilitate extensive studies of host and organism dynamics during experimental pharyngitis. Ethics approval has been obtained and the study has been registered at ClinicalTrials.gov (NCT03361163)., info:eu-repo/semantics/published

Purpose There is a need to broaden protective coverage of M protein-based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. Materials and methods A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. Results The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein-based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. Conclusion These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines.

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.

Protein subunit vaccines are often preferred because of their protective efficacy and safety. Lactic acid bacteria expressing heterologous antigens constitute a promising approach to vaccine development. However, their safety in terms of toxicity and bacterial clearance must be evaluated. Anti-Streptococcus pyogenes (S. pyogenes) vaccines face additional safety concerns because they may elicit autoimmune responses. The assessment of toxicity, clearance and autoimmunity of an anti-streptococcal vaccine based on Lactococcus lactis (L. lactis) expressing 10 different M protein fragments from S. pyogenes (L. lactis-Mx10) is here reported. Clearance of L. lactis from the oropharynges of immunocompetent mice and mice devoid of T/B lymphocytes mice was achieved without using antibiotics. The absence of autoimmune responses against human tissues was demonstrated with human brain, heart and kidney. Assessment of toxicity showed that leucocyte counts and selected serum biochemical factors were not affected in L. lactis-Mx10-immunized mice. In contrast, mice immunized with L. lactis wild type vector (L. lactis-WT) showed increased neutrophil and monocyte counts and altered histopathology of lymph nodes, lungs and nasal epithelium. Two days after immunization, L. lactis-Mx10-immunized and L. lactis-WT-immunized mice weighed significantly less than unimmunized mice. However, both groups of immunized mice recovered their body weights by Day 6. Our results demonstrate that L. lactis-WT, but not the vaccine L. lactis-Mx10, induces alterations in certain hematologic and histopathological variables. We consider these data a major contribution to data on L. lactis as a bacterial vector for vaccine delivery.

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N-terminal fragment of M protein, has been developed. Live bacterial-vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10-valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three- to 10-fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10-immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10-immunized mice did not differ significantly from that of unimmunized mice. Mx-10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

Group A Streptococcus (GAS) skin infections are particularly prevalent in developing nations. The GAS M protein, by which strains are differentiated into >220 different emm types, is immunogenic and elicits protective antibodies. A major obstacle for vaccine development has been the traditional understanding that immunity following infection is restricted to a single emm type. However, recent evidence has led to the hypothesis of immune cross-reactivity between emm types., SCOPUS: ar.j, info:eu-repo/semantics/published

Influenza virus infections are associated with a significant number of illnesses and deaths on an annual basis. Many of the deaths are due to complications from secondary bacterial invaders, including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pyogenes. The β-hemolytic bacteria S. pyogenes colonizes both skin and respiratory surfaces, and frequently presents clinically as strep throat or impetigo. However, when these bacteria gain access to normally sterile sites, they can cause deadly diseases including sepsis, necrotizing fasciitis, and pneumonia. We previously developed a model of influenza virus:S. pyogenes super-infection, which we used to demonstrate that vaccination against influenza virus can limit deaths associated with a secondary bacterial infection, but this protection was not complete. In the current study, we evaluated the efficacy of a vaccine that targets the M protein of S. pyogenes to determine whether immunity toward the bacteria alone would allow the host to survive an influenza virus:S. pyogenes super-infection. Our data demonstrate that vaccination against the M protein induces IgG antibodies, in particular those of the IgG1 and IgG2a isotypes, and that these antibodies can interact with macrophages. Ultimately, this vaccine-induced immunity eliminated death within our influenza virus:S. pyogenes super-infection model, despite the fact that all M protein-vaccinated mice showed signs of illness following influenza virus inoculation. These findings identify immunity against bacteria as an important component of protection against influenza virus:bacteria super-infection.

Background The greatest burden of group A streptococcal (GAS) disease worldwide is due to acute rheumatic fever (ARF) and rheumatic heart disease (RHD). Safe, effective and affordable vaccines designed to prevent GAS infections that trigger ARF could reduce the overall global morbidity and mortality from RHD. The current study evaluated the potential coverage of a new 30-valent M protein-based vaccine using GAS isolates from school children in Bamako, Mali, a population at high risk for the development of RHD. Methods The bactericidal activity of rabbit antisera against the 30-valent vaccine was assessed using a collection of GAS isolates recovered during a study of the epidemiology of pharyngitis in Bamako. Results Single isolates representing 42 of 67 emm -types, accounting for 85% of the GAS infections during the study, were evaluated. All (14/14) of the vaccine emm -types in the collection were opsonized (bactericidal killing >50%) and 26/28 non-vaccine types were opsonized. Bactericidal activity was observed against 60% of the total emm -types recovered in Bamako, which accounted for 81% of all infections. Conclusions Multivalent vaccines comprised of N-terminal M peptides elicit bactericidal antibodies against a broad range of GAS serotypes, indicating that their efficacy may extend beyond the emm -types included in the vaccine.

Background. Despite the significant burden of disease associated with infection by group A streptococcus (GAS), little is known about the human immune response to GAS antigens after natural infection. Methods. We evaluated 195 serum samples obtained prospectively over a consecutive 24-month period from 41 pediatric subjects who experienced a new pharyngeal GAS acquisition. An enzyme-linked immunoassay was used to determine the kinetics and antigen specificity of antibodies against 13 shared GAS antigens and 18 type-specific M peptides. The majority of the antigens tested are currently being considered as vaccine candidates. Results. Twelve M types of GAS were recovered from 41 subjects who experienced 51 new GAS acquisitions that elicited antibody responses against at least 1 of the 31 antigens tested (immunologically significant new GAS acquisitions). The immune responses to the 13 shared antigens were highly variable. Increases in antibody levels were detected against a mean of 3.5 shared antigens (range, 1–8). Antibody responses to the homologous M peptide were observed in 32 (63%) of the 51 episodes. Seven subjects acquired more than 1 M type of GAS. There were no new immunologically significant acquisitions of an M type against which the subject had preexisting antibodies to the homologous M peptide. Of the subjects with new GAS acquisition, 65% were asymptomatic, yet immune responses were detected against 1 or more GAS antigens. Immune responses to streptolysin O and/or deoxyribonuclease B were observed after 67% of the new GAS acquisitions. Persistently positive (>12 weeks) throat culture results were returned for 20% of the 41 subjects despite immune responses to homologous M peptides and/or shared antigens. Conclusions. The availability of throat culture results, GAS isolates, and serial serum samples collected prospectively over a 2-year period of observation provided a unique opportunity for us to assess the serologic status of pediatric subjects before and after new pharyngeal acquisitions of GAS. With the exception of antibody responses to the homologous M peptides, no clear pattern of immune responses against the remaining GAS antigens was seen. There were no new immunologically significant acquisitions ofemm types of GAS against which the subjects had preexisting elevated levels of antibodies against the homologous M peptide. The observation that 65% of new GAS acquisitions caused no symptoms yet were immunologically significant suggests that the majority of infections are not detected, which would result in missed opportunities for primary prevention of rheumatic fever and rheumatic heart disease with appropriate antimicrobial therapy.

Background A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. Methods M protein sequences (AA 16–50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4G AS. Results The synthetic vaccine rabbit antisera reacted with all 17 E4 M peptides and demonstrated bactericidal activity against 15/17 E4G AS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4 M peptides. Conclusions Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS.

The ability to hide in the animal kingdom is essential for survival; the same is true for bacteria. Streptococcus pyogenes is considered one of the more successful stealth bacteria in its production of a hyaluronic acid capsule that is chemically identical to the hyaluronic acid lining human joints. It has also acquired the capacity to enter eukaryotic cells to avoid the onslaught of the host’s immune defenses, as well as drugs. From this intracellular vantage point, it may remain dormant from days to weeks, only to cause disease again at a later time, perhaps causing a relapse in a drug-treated patient. We now learn that it is able to enter macrophages as well, enabling the Streptococcus to use this “Trojan horse” approach to be transported to distant sites in the body.

Our previous studies have shown that recombinant multivalent vaccines containing amino-terminal M protein fragments from as many as 26 different serotypes of group A streptococci (GAS) evoked opsonic antibodies in animals and humans. In the present study, we constructed a new 30-valent vaccine containing M protein peptides from GAS serotypes prevalent in North America and Europe. The vaccine was immunogenic in rabbits and evoked bactericidal antibodies against all 30 vaccine serotypes of GAS. In addition, the vaccine antisera also contained significant levels of bactericidal antibodies against 24 of 40 non-vaccine serotypes of GAS. These results indicate that the potential efficacy of the new multivalent vaccine may be greater than predicted based on the “type-specific” M peptides represented.

BACKGROUND The NH(2)-terminal sequence of the M protein from group A streptococci defines the serotype of the organism and contains epitopes that evoke bactericidal antibodies. METHODS To identify additional roles for this region of the M protein, we constructed a mutant of M5 group A streptococci expressing an M protein with a deletion of amino acid residues 3-22 (DeltaNH(2)). RESULTS M5 streptococci and the DeltaNH(2) mutant were resistant to phagocytosis and were similarly virulent in mice. However, DeltaNH(2) was significantly less hydrophobic, contained less lipoteichoic acid on its surface, and demonstrated reduced adherence to epithelial cells. These differences were abolished when organisms were grown in the presence of protease inhibitors. Treatment with cysteine proteases or with human saliva resulted in the release of M protein from the DeltaNH(2) mutant at a significantly greater rate than observed with the wild-type M5 strain. Compared with the parent strain, the DeltaNH(2) strain also showed a significant reduction in its ability to colonize the upper respiratory mucosa of mice. CONCLUSIONS The NH(2) terminus of M5 protein has an important role in protecting the surface protein from proteolytic cleavage, thus preserving its function as an anchor for lipoteichoic acid, which is a primary mediator of adherence to epithelial cells and colonization.

Our prior studies of the molecular epidemiology of group A streptococcus (GAS) pharyngitis indicated that the most common emm types associated with pediatric pharyngitis in North America were 12, 1, 28, and 4. We previously reported that the proportions of pediatric pharyngitis due to emm types 12 and 4 decreased with increasing age throughout childhood. We hypothesized that this is due to age-associated acquisition of antibodies to the amino-terminal type-specific region of common GAS M proteins during childhood. We sought to demonstrate this in sera from healthy children by using ELISAs for M 12, 1, 28, and 4. Enzyme-linked immunosorbent assays (ELISA) using chemically synthesized peptides copying amino-terminal type-specific regions of the M proteins were performed on sera from four age groups of healthy children (group I: 3-6 years, group II: 7-10 years, group III: 11-14 years, group IV: 15-18 years). ELISA data were correlated with opsonophagocytic assays for a subset of sera and M 1 GAS. Sera from healthy 12-20-month-old children were used as negative controls. Our results showed that the highest percentage of positivity was for M12, which also showed progressive seropositivity in older children. For the other serotypes, the highest seroprevalence rates were in the 11-14-year-old age group. The presence of ELISA antibodies against M1 correlated with opsonophagocytic activity, a previously studied indicator of immunologic protection.

Introduction Group A β-haemolytic Streptococcus (GAS), a Gram-positive bacterium, also known as Streptococcus pyogenes , causes pyoderma, pharyngitis and invasive disease. Repeated GAS infections may lead to autoimmune diseases such as acute post-streptococcal glomerulonephritis, acute rheumatic fever (ARF) and rheumatic heart disease (RHD). Invasive GAS (iGAS) disease is an important cause of mortality and morbidity worldwide. The burden of GAS infections is, however, unknown in Africa because of lack of surveillance systems. Methods and analysis The African group A streptococcal infection registry (the AFRO Strep study) is a collaborative multicentre study of clinical, microbiological, epidemiological and molecular characteristics for GAS infection in Africa. The AFRO Strep registry comprises two components: (1) active surveillance of GAS pharyngitis cases from sentinel primary care centres (non-iGAS) and (2) passive surveillance of iGAS disease from microbiology laboratories. Isolates will also be subjected to DNA isolation to allow for characterisation by molecular methods and cryopreservation for long-term storage. The AFRO Strep study seeks to collect comprehensive data on GAS isolates in Africa. The biorepository will serve as a platform for vaccine development in Africa. Ethics and dissemination Ethics approval for the AFRO Strep registry has been obtained from the Human Research Ethics Committee at the University of Cape Town (HREC/REF: R006/2015). Each recruiting site will seek ethics approval from their local ethics’ committee. All participants will be required to provide consent for inclusion into the registry as well as for the storage of isolates and molecular investigations to be conducted thereon. Strict confidentiality will be applied throughout. Findings and updates will be disseminated to collaborators, researchers, health planners and colleagues through peer-reviewed journal articles, conference publications and proceedings.

Macrolide resistance (MR) among Group A streptococci (GAS) has implications for treatment of pharyngeal and invasive infections. In Year 1 (2000–2001) and Year 2 (2001–2002) of our ongoing surveillance of pharyngeal GAS, MR rates in the United States were 4.4% and 4.3%, respectively. We now report MR rates among GAS in the US and Canada and describe national differences in emm types and MR genotypes for 2002–2003 (Year 3), 2003–2004 (Year 4), and 2004–2005 (Year 5). MR rates were higher each year in Canada than in the US but the rates appear to be converging. Resistance rates increased significantly in the US from 3.8% in Year 3 to 8.4% in Year 5. MR genotypes and the most prevalent MR emm types varied significantly between the two neighbor nations.

M type 4 Streptococcus pyogenes is a frequent cause of infections, yet little is known concerning its anti-phagocytic factors. We investigated the role of the Mga regulon on growth in blood by inactivating individual genes within this regulon. Ablation of mrp4, emm4, or sof4 reduced streptococcal growth in human blood. Inactivation of enn4 had little impact on streptococcal growth in blood. Thus, expression of mrp, emm, and sof were required for optimal resistance to phagocytosis of M type 4 S. pyogenes. M proteins from class II serotypes contribute to resistance to phagocytosis by binding C4BP, a regulator of complement activation. Our data indicate that there is an additional pathway for resistance in these streptococci that is mediated by the binding of fibrinogen. Fibrinogen was required for growth of M type 4 S. pyogenes in blood and Mrp4 was the major fibrinogen-binding protein on these bacteria. Fibrinogen–Mrp interactions prevented activation of the classical pathway, thereby contributing to the ability of these streptococci to evade phagocytosis.

The geographic and year–year heterogeneity of group A streptococcal (GAS) genotypes (emm types) of isolates causing acute pediatric pharyngitis in North America have been poorly characterized. We established a network of 10 U.S. and 3 Canadian sites to collect isolates from children 3–18 years old with acute streptococcal pharyngitis. Collection began in 2000–2001, and the fifth year of collection was completed in May 2005. Multiple GAS types circulated simultaneously within each study site. Each year the 3 most prevalent emm types in the U.S. or Canada comprised 42–49% of all isolates nationally. The 6 most prevalent types each year consistently comprised 70–77% of isolates. Analysis of data from individual sites demonstrated considerable (in some instances striking) geographic variability as well as year–year variability. Differences were identified between the distribution of emm types from U.S. and Canadian sites. Continued pharyngeal emm type surveillance will be needed as multivalent M protein vaccine development proceeds.

Background GrAS causes illness ranging from uncomplicated pharyngitis to life-threatening necrotizing fasciitis, toxic shock and rheumatic fever. Attempts to develop an M protein-based vaccine have been hindered by the fact that some M proteins elicit both protective antibodies and antibodies that cross-react with human tissues. New molecular techniques have allowed this obstacle to be largely overcome. Methods We performed a phase II trial of a 26-valent GrAS vaccine comprising four recombinant proteins containing N-terminal peptides from 26 M proteins (plus Spa = 27) of common pharyngitis, invasive, and/or rheumatogenic serotypes adsorbed to AlOH 3 . Subjects were screened for good health and underwent baseline cardiac auscultation, echocardiography, ECG, and screening for human tissue-cross-reacting antibodies. Subjects (mean age = 33.6, range 18.9–50.9) were randomized in a 70:20 ratio to receive either GrAS or control vaccine, Havrix™, intramuscularly at 0, 1 and 6 months, with clinical and laboratory follow-up for safety and assay of type-specific M antibodies by quantitative ELISA. Results No vaccine-associated serious adverse events (SAE) occurred. Most adverse events (AEs) were at the injection site and were mild and self-limited. Systemic AEs were uncommon and did not differ between the 2 groups. No subject developed clinical or laboratory evidence of rheumatogenicity or nephritogenicity, and no induction of human tissue-reactive antibodies was demonstrated. The vaccine was highly immunogenic, eliciting an antibody response to the majority of group A streptococcus serotypes associated with pharyngitis, acute rheumatic fever, and invasive disease in North America Conclusions The 26-valent GrAS vaccine was well-tolerated and immunogenic in healthy adults; studies in adolescents and children are warranted.

A key attribute of invasive Streptococcus pyogenes is their ability to resist phagocytosis and multiply in human blood. M-related protein (Mrp) is a major anti-phagocytic factor but the mechanism whereby it helps streptococci to evade phagocytosis has not been demonstrated. We investigated phagocytosis resistance in a strain of serotype M4 by inactivating the mrp gene and also the emm, enn, sof and sfbX genes and by analysing the effect on streptococcal growth in blood and on complement deposition on the bacterial surface. Inactivation of enn4 and sfbX4 had little impact on growth in blood, but ablation of mrp4, emm4 or sof4 reduced streptococcal growth in human blood, confirming that Mrp and Emm are required for optimal resistance to phagocytosis and providing the first indication that Sof may be an anti-phagocytic factor. Moreover, antisera against Mrp4, Emm4 and Sof4 promoted the killing of S. pyogenes, but anti-SfbX serum had no effect. Growth of S. pyogenes in blood was dependent on the presence of fibrinogen and in the absence of fibrinogen there was a twofold increase in complement deposition. Inactivation of mrp4 resulted in a loss of fibrinogen-binding and caused a twofold increase in the binding of C3b that was inhibited by Mg-EGTA. Mrp contained two fibrinogen-binding sites, one of which is within a highly conserved region. These findings indicate that Mrp-fibrinogen interactions prevent surface deposition of complement via the classical pathway, thereby contributing to the ability of these streptococci to resist phagocytosis. This may be a common mechanism for evasion of phagocytosis because Mrp is expressed by approximately half of the clinical isolates of S. pyogenes.

The molecular epidemiology of group A streptococcal pharyngeal infections in children in the Vanguard Community of Cape Town revealed 26 emm types among 157 isolates from 742 subjects. Coverage of a 30-valent vaccine is predicted to be 95% of pharyngitis cases in this population at high risk of rheumatic fever.

Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.

Geographic and interseasonal heterogeneity of pharyngeal group A streptococcal (GAS) genotypes (emm types) is poorly characterized. We evaluated emm type and subtype distribution among pediatric pharyngitis isolates obtained from 9 sites in the United States during 2000-2001 (year 1) and from 10 sites in the United States and 1 site in Canada during 2001-2002 (year 2). The 7 predominant types were the same in both years, although their order changed. emm 12, 1, and 28 accounted for 49.2% of year 1 isolates, and emm 1, 12, and 4 accounted for 47.1% of year 2 isolates; 6 types accounted for 72.1% in year 1 and 69.4% in year 2. From year 1 to year 2, the proportions of emm 12 and 28 decreased and emm 1 and 6 increased. Striking intersite and interseasonal variations in the distribution of predominant emm types were observed. We conclude that the most-predominant GAS genotypes were similar for each year despite fluctuations, that intersite and intrasite variations in the distribution of emm types were apparent, and that emm type surveillance is needed as M protein vaccine development proceeds.

Serum opacity factor (SOF) is a protein expressed byStreptococcus pyogenesthat opacifies mammalian serum. SOF is also a virulence factor ofS. pyogenes, but it has not been previously shown to elicit a protective immune response. Herein, we report that SOF evokes bactericidal antibodies againstS. pyogenesin humans, rabbits, and mice. Rabbit antiserum against purified recombinant SOF2 opsonized SOF-positive M type 2, 4, and 28S. pyogenesin human blood but had no effect on SOF-negative M type 5S. pyogenes. Furthermore, affinity-purified human antibodies against SOF2 also opsonized SOF-positive streptococci. A combination of antisera against M2 and SOF2 proteins was dramatically more effective in killing streptococci than either antiserum alone, indicating that antibodies against SOF2 enhance the opsonic efficiency of M protein antibodies. Mice tolerated an intravenous injection of 100 μg of SOF without overt signs of toxicity, and immunization with SOF protected mice against challenge infections with M type 2S. pyogenes. These data indicate that SOF evokes opsonic antibodies that may protect against infections by SOF-positive serotypes of group A streptococci and suggest that different serotypes of SOF have common epitopes that may be useful vaccine candidates to protect against group A streptococcal infections.

Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33mrpgenes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GASemmtypes expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing differentemmtypes that expressed an Mrp within the same family but showed no activity againstemmtypes expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines.