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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].

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.

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.

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.

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.

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.

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.

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.

Serum opacity factor (SOF) is a large, extracellular, and cell-bound protein of group A streptococci that has two known functions, opacification of serum and binding of fibronectin. Herein, we describe a new function of SOF, the binding of fibrinogen. Utilizing purified, truncated recombinant SOF proteins, the fibrinogen-binding domain was localized to a region in the C-terminus of SOF encompassing amino acid residues 844-1047. Western-blot analysis revealed that SOF bound primarily to the beta subunit of fibrinogen. A SOF-negative mutant bound 50% less fibrinogen than did its wild-type parent. Furthermore, fibrinogen blocked the binding of SOF to fibronectin. These data suggest that fibrinogen and fibronectin bind to the same domain within SOF. It remains to be determined whether the binding of fibrinogen to SOF contributes to the virulence of group A streptococci.

A multivalent vaccine containing amino-terminal M protein fragments from 26 different serotypes of group A streptococci was constructed by recombinant techniques. The vaccine consisted of four different recombinant proteins that were formulated with alum to contain 400 μg of protein per dose. Rabbits were immunized via the intramuscular route at 0, 4, and 16 weeks. Immune sera were assayed for the presence of type-specific antibodies against the individual recombinant M peptides by enzyme-linked immunosorbent assay and for opsonic antibodies by in vitro opsonization tests and indirect bactericidal tests. The 26-valent vaccine was highly immunogenic and elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. The immune sera were broadly opsonic and were bactericidal against the majority of the 26 different serotypes. Importantly, none of the immune sera cross-reacted with human tissues. Our results indicate that type-specific, protective M protein epitopes can be incorporated into complex, multivalent vaccines designed to elicit broadly protective opsonic antibodies in the absence of tissue-cross-reactive antibodies.

BACKGROUND Group A streptococcus (GAS) pharyngitis is associated with high rates of rheumatic heart disease in developing countries. We sought to identify guidelines for empiric treatment of pharyngitis in low-resource settings. To inform the design of GAS vaccines, we determined the emm types associated with pharyngitis among African schoolchildren. METHODS Surveillance for pharyngitis was conducted among children 5-16 years of age attending schools in Bamako, Mali. Students were encouraged to visit a study clinician when they had a sore throat. Enrollees underwent evaluation and throat swab for isolation of GAS. Strains were emm typed by standard methods. RESULTS GAS was isolated from 449 (25.5%) of the 1,759 sore throat episodes. Painful cervical adenopathy was identified in 403 children (89.8%) with GAS infection and was absent in 369 uninfected children (28.2%). Emm type was determined in 396 (88.2%) of the 449 culture-positive children; 70 types were represented and 14 types accounted for 49% of isolates. Based on the proportion of the 449 isolates bearing emm types included in the 30-valent vaccine (31.0%) plus nonvaccine types previously shown to react to vaccine-induced bactericidal antibodies (44.1%), the vaccine could protect against almost 75% of GAS infections among Bamako schoolchildren. CONCLUSIONS Two promising strategies could reduce rheumatic heart disease in low-resource settings. Administering antibiotics to children with sore throat and tender cervical adenopathy could treat most GAS-positive children while reducing use of unnecessary antibiotics for uninfected children. Broad coverage against M types associated with pharyngitis in Bamako schoolchildren might be achieved with the 30-valent GAS vaccine under development.

Group A streptococci (GAS) ( Streptococcus pyogenes ) are common causes of infections in humans for which there is no licensed vaccine. Decades of work has focused on the role of the surface M protein in eliciting type-specific protective immunity. Recent studies have identified additional surface proteins of GAS that contain opsonic epitopes. In the present study, we describe a serotype M65 GAS originally isolated during an epidemiologic study in Bamako, Mali, which simultaneously expressed M, M-related protein (Mrp), and streptococcal protective antigen (Spa) on the bacterial surface. The emm , mrp , and spa genes were sequenced from PCR amplicons derived from the M65 chromosome. Rabbit antisera raised against synthetic peptides copying the N-terminal regions of M, Mrp, and Spa were highly specific for each peptide, reacted with the surface of M65 GAS, and promoted bactericidal activity against the organism. A mixture of antisera against all three peptides was most effective in the bactericidal assays. Immunofluorescence microscopy revealed that the M, Mrp, and Spa antisera bound to the bacterial surface in the presence of human plasma proteins and resulted in the deposition of complement. Five additional spa genes were identified in the Mrp-positive GAS serotypes, and their sequences were determined. Our results indicate that there are multiple antigens on the surface of GAS that evoke antibodies that promote bacterial killing. A more complete understanding of the relative contributions of M, Mrp, and Spa in eliciting protective immunity may aid in the development of GAS vaccines with enhanced coverage and efficacy.

It is widely believed that the surface M protein of group A streptococci is the predominant surface protein of these organisms containing opsonic epitopes. In the present study, we identified a new surface protein, distinct from M protein, that evokes protective antibodies. A type 18 M-negative mutant was found to be both resistant to phagocytosis in human blood and virulent in mice. The wild-type strain, but not the M-negative mutant, was opsonized by antisera against purified recombinant M18 protein or a synthetic peptide copying the NH2-terminus of M18. However, antisera raised against a crude pepsin extract of the M-negative mutant opsonized both strains, indicating the presence of a protective antigen in addition to type 18 M protein. This antiserum was used to identify and purify a 24-kDa protein fragment (Spa, streptococcal protective antigen) that evoked antibodies that opsonized the M18 parent and the M-negative mutant. The results of passive mouse protection tests confirmed the presence of protective epitopes within Spa. The deduced amino acid sequence of a 636-bp 5′ fragment of the spa18 gene showed no homology with sequences in GenBank. These studies reveal the presence of a new protective antigen of certain strains of group A streptococci that may prove to be an important component of vaccines to prevent streptococcal infections.

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.

The present invention provides methods for eliciting an immune response against Group A streptococci, comprising use of recombinant fusion polypeptides, and compositions thereof, that include a multivalent immunogenic portion of at least two immunogenic polypeptides from Group A streptococci M proteins (which are capable of stimulating a protective immune response against Group A streptococci), and a reiterated polypeptide from the immunogenic portion carboxy-terminal to the immunogenic portion, wherein the carboxy-terminal polypeptide is not required to stimulate an immune response against Group A streptococci.

One of the major challenges in the development of group A streptococcal M protein-based vaccines is the multiplicity of M types expressed by these organisms. Previous studies have shown that multivalent vaccines containing as many as eight M protein fragments in tandem were immunogenic and evoked opsonic antibodies. It was also noted that the C-terminal fragments of these hybrid proteins were often not immunogenic or evoked only low levels of opsonic antibodies, suggesting that the C-terminus of the molecule may have been preferentially degraded or altered in vivo. In the present studies, we designed a hexavalent vaccine containing protective M protein peptides from types 24, 5, 6, 19, 1, and 3 group A streptococci. In order to "protect" the carboxy-terminal components, the amino-terminal M24 fragment was reiterated on the carboxy-terminal end of the construct. The hexavalent vaccine was immunogenic in laboratory animals and evoked high titers of antibodies against each of the native M proteins represented in the vaccine and bactericidal antibodies against all six sterotypes of group A streptococci. The vaccine was equally immunogenic when delivered in alum or in complete Freund's adjuvant. None of the immune sera contained antibodies that crossreacted with human heart tissue. Our results show that complex multivalent group A streptococcal vaccines can be designed in such a way that each M protein fragment is immunogenic and evokes protective antibodies.

The availability of a protective vaccine againstStreptococcus pyogenes(group AStreptococcus[GAS]) is a priority for public health worldwide. Here, we have generated six live vaccine strains, each engineered to express an N-terminal M protein peptide from one of six of the most prevalentemmtypes of GAS (M1, M2, M4, M9, M12, and M28). The vaccine strains are based on a food-gradeLactococcus lactisstrain and do not bear any antibiotic resistance. Mice immunized with the vaccine strain expressing the M9 peptide (termed here theL. lactisM9 strain) showed high titers of serum antibodies when delivered intranasally. Mice immunized with theL. lactisM9 strain were protected against infection after intranasal challenge with type 9 streptococci. Several parameters of disease, such as weight loss, body temperature, colony counts in mouth washes, and lung histology, were significantly improved in immunized mice compared to naive control mice. Our results indicate that intranasal delivery of theL. lactisM9 strain live bacterial vaccine induced GAS-specific IgG titers, prevented pharyngeal colonization of GAS, and protected mice from disease upon challenge. The design of this vaccine prototype may provide a lower cost alternative to vaccines comprised of purified recombinant proteins.

One of the major obstacles to the development of group A streptococcal M protein vaccines is the multiplicity of M serotypes expressed by these organisms. In this study, we have constructed a recombinant, hybrid M protein that contains type-specific aminoterminal fragments of eight different M proteins. We show that the purified hybrid recombinant protein is immunogenic in rabbits and evokes antibodies that react with native M proteins from the respective streptococcal serotypes. In addition, the immune sera evoked by the octavalent protein opsonized six of the eight serotypes of streptococci, indicating that the majority of the M protein fragments contained protective epitopes that retained their native conformations in the hybrid protein. None of the antisera raised against the octavalent protein crossreacted with human heart tissue. These studies indicate that multivalent, hybrid M proteins may be used to elicit broadly protective immune responses against multiple serotypes of group A streptococci.

The major virulence determinant of group A streptococci is the ability to resist opsonization and phagocytic ingestion. The present studies were performed to compare the mechanisms of resistance to opsonization of type 18 and type 24 streptococci and to determine the relative roles of M protein-fibrinogen interaction and the hyaluronate capsule in preventing phagocytic ingestion and killing. By use of parent strains and acapsular transposon mutants in the presence and absence of fibrinogen, we show that type 18 and type 24 streptococci rely on somewhat different mechanisms for resistance to opsonization. Type 24 streptococci bound fibrinogen avidly to their surfaces, and encapsulated organisms were completely resistant to opsonization only in the presence of fibrinogen. In contrast, type 18 streptococci bound 10-fold less fibrinogen than type 24 streptococci and were fully resistant to phagocytosis only when they expressed capsule. The general structural characteristics of the amino-terminal halves of type 18 and type 24 M proteins differed in that type 18 M protein contained only one complete B repeat, whereas type 24 M protein contained five complete B repeats, a structural difference which could potentially be related to the differences in fibrinogen binding between the two serotypes. Immunofluorescence assays of complement deposition were used in combination with 125I-C3 binding assays to show that encapsulated type 24 streptococci were fully resistant to opsonization by C3 only in the presence of plasma. Encapsulated and unencapsulated type 18 streptococci were equally opsonized by C3 in either plasma or serum, yet only encapsulated organisms resisted phagocytic killing in blood. The results of this study indicate that opsonization by C3 does not necessarily lead to phagocytic ingestion and that the hyaluronate capsule and M proteins are variably important in resistance to different group A streptococci to opsonization and phagocytic killing.

Group A streptococci (GAS) are important causes of morbidity and mortality worldwide. These organisms cause a wide spectrum of disease, ranging from uncomplicated sore throat to invasive, life-threatening infections, as well as immune complications such as acute rheumatic fever (ARF), rheumatic heart disease (RHD) and acute post-streptococcal glomerulonephritis (APSGN). Vaccine prevention of GAS infections and their immunological complications has been a goal of researchers for decades. Several vaccine candidates against GAS infection are in various stages of pre-clinical and clinical development, including M protein-based vaccines (N-terminal vaccine candidates and M protein conserved region vaccines), and non-M protein vaccine candidates representing conserved GAS antigens. Some of the obstacles to GAS vaccine development are related to the complexity of the global epidemiology of GAS infections, the limitation in the criteria for selection of antigens to include in combination vaccines as well as the issues around autoimmunity and vaccine safety, among others. Overcoming these obstacles will require collaborative efforts to develop innovative strategies that address key steps in the pre-clinical and clinical development process, as well as clearly defining the global burden of GAS diseases and the molecular epidemiology of infections. Specific recommendations are presented for an accelerated plan leading to the introduction of a broadly protective vaccine designed for deployment in low-, middle-, and high-income countries.

Lipoteichoic acid and several streptococcal proteins have been reported to bind fibronectin (Fn) or fibrinogen (Fgn), which may serve as host receptors. We searched for such proteins by screening a library of genes from M type 5 group A streptococci cloned into Escherichia coli. Lysates of clones were probed with biotinylated Fn and biotinylated Fgn. One clone expressed a 54-kDa protein that reacted with Fn and Fgn. The protein, termed FBP54, was purified and used to immunize rabbits. Anti-FBP54 serum reacted with purified, recombinant FBP54 and with a protein of similar electrophoretic mobility in extracts of M type 5, 6, and 24 streptococci. Anti-FBP54 serum also reacted with 5 of 15 strains of intact, live streptococci, suggesting that FBP54 may be a surface antigen. Southern blot analysis confirmed that the gene is found in group A streptococci but not in Staphylococcus aureus or E. coli. The cloned gene was sequenced and contained an open reading frame encoding a protein with a calculated molecular weight of 54,186. Partial amino acid sequencing of purified FBP54 confirmed that this open reading frame encoded the protein. As determined by utilizing fusion proteins containing truncated forms of FBP54, the primary Fn/Fgn-binding domain appears to be contained in residues 1 to 89. These data suggest that FBP54 may be a surface protein of streptococci that reacts with both Fn and Fgn and therefore may participate in the adhesion of group A streptococci to host cells.

To the Editor—A new emm-cluster typing system has been recently proposed for group A Streptococcus (GAS) [1]. This system classifies most of the 223 emm types [2] into 48 functional emm clusters containing closely related M proteins that share structural properties. emm clusters help to predict the virulence potential of any GAS isolate by ascribing M protein binding attributes to emm types belonging to the same emm cluster [1, 3]. This system correlates with M protein vaccine antigen content and serves as a framework to investigate immunologic cross-protection between emm types [1, 4, 5]. emm clusters have been used to analyze the epidemiology of GAS in the Pacific region, which is characterized by high GAS disease burden and a great variety of circulating emm types [6, 7]. The emm-cluster system identified epidemiologic similarities across the Pacific region and highlighted vaccine target priorities [8]. We applied the emm-cluster system to GAS epidemiology in a high-income setting by analyzing prospective surveillance data for GAS pharyngitis in North America, 2000–2007 [9]. A total of 56 and 33 different emm types were recovered from 7040 US and 1434 Canadian GAS isolates, respectively. In contrast with the Pacific region, the 15 most prevalent emm types accounted for 97.1% and 96.9% of GAS pharyngeal isolates, respectively, indicating that only a relative minority of emm types are responsible for most pharyngitis in North America. By deducing the emm-cluster allocation from the emm-typing results [1], we observed that the 56 US emm types belonged to 18 emm clusters (Table ​(Table1),1), whereas the 33 Canadian emm types belonged to 14 emm clusters (data not shown). Eleven emm clusters were responsible for the majority of cases in both countries (99.6% and 98.7%, respectively) (Table ​(Table1).1). emm types 1 and 12 were the 2 most common emm types in the United States (17.8% and 17.6%, respectively) [9], but did not belong to the most common emm cluster; rather, emm cluster E4 (notably including the frequent emm types 2, 22, 28, 77, and 89) was more common (27.2%). Furthermore, we observed that 8 of the 56 emm types belonged to emm cluster D4, although representing only a small number of isolates (17 of 7040), a surprising result given that emm cluster D4 is associated with skin rather than pharyngeal infections [1]. Finally, emm cluster E2, which includes 15 emm types, was nearly completely absent from North America (0.2%–0.5% of isolates), whereas it was the most frequent emm cluster in New Caledonia (21% of isolates), suggesting that some emm clusters are restricted to defined geographical areas. Table 1. Frequencies of emm Types and emm Clusters Among 7040 Group A Streptococcus Isolates Recovered From Pharyngitis in the United States As shown here, and in the study in New Caledonia, application of the emm-cluster system to both tropical and nontropical settings improves our understanding of complex GAS epidemiology. This new system helps to refine clinically meaningful questions such as tissue tropism and the immune response to GAS infections in all settings worldwide.

The amino terminal region of group A streptococcal M proteins evokes type-specific immunity while the conserved C-repeat epitopes evoke cross-protective immunity against multiple serotypes. The present studies were undertaken to compare the protective efficacy of vaccines containing either type-specific (hexavalent vaccine) or conserved C-repeat (J14 vaccine) M protein epitopes and to determine if combination vaccines resulted in enhanced levels of protection. Our results indicated that the protective efficacy of the type-specific vaccine was significantly greater than that of J14 and that the addition of J14 to vaccine formulations did not enhance the level of protection achieved with type-specific formulations.

BACKGROUND Pharyngeal group A streptococcal (GAS) emm type surveillance enhances understanding of the epidemiology of pharyngitis and invasive GAS disease and formulation of multivalent type-specific vaccines. In addition, such surveillance provides pre-GAS vaccine baseline data. We assessed geographic and temporal trends in GAS emm-type distribution among pediatric pharyngeal isolates collected systematically in the United States and Canada from 2000 to 2007. METHODS We collected approximately 100 acute GAS pharyngitis isolates from each of 13 widely scattered sites (10 in the United States and 3 in Canada) annually for 7 seasons (2000-2007) from 3- to 18-year-old children. We assessed emm type and subtype by DNA sequencing and analyzed temporal and geographic trends. RESULTS A total of 7040 US and 1434 Canadian GAS isolates were studied. The 6 most prevalent emm types (in descending order) were 1, 12, 28, 4, 3, and 2 in the United States and 12, 1, 28, 4, 3, 2, and 77 in Canada, constituting 70%-71% of isolates in each country; 10 emm types constituted 87%-89% total. Fifty-six emm types were identified in the United States, including 8 new types, and 33 types in Canada. Although a few types predominated nationally, marked variability among individual sites and at individual sites from year to year was observed. US-Canadian differences in type distribution were apparent. Twenty percent of isolates represented emm subtypes that differed slightly from reference types; 110 new subtypes were identified. An experimental 26-valent M protein vaccine covers 85% of pharyngitis isolates. CONCLUSIONS Although overall US and Canadian emm type distribution was consistent and relatively few types dominated nationally, striking intersite and temporal variations within individual sites in prevalent emm types of GAS occurred. These results have important implications for the development and formulation of type-specific GAS vaccines.

Previous studies in our laboratory described a new group A streptococcal protective antigen (Spa) in type 18 streptococci that was distinct from the type 18 M protein. This study was undertaken to identify additional serotypes of group A streptococci that express Spa proteins. PCR techniques were used to identify and clone a new spa gene from type 36 streptococci. The 5′ sequence of spa36 was highly variable compared to spa18, while the 3′ sequence was conserved. Antisera against Spa36 opsonized type 36 streptococci but not type 18 streptococci, indicating that the opsonic Spa epitopes were type-specific. Antisera against the conserved carboxy-terminal half of Spa18 were used to identify Spa or Spa-like proteins expressed on the surface of 25 of 70 different serotypes of GAS. Spa proteins may represent a new family of type-specific surface antigens that function in concert with M proteins to elicit protective immune responses.