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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A fusion gene named LT-B-M5 was constructed encoding the entire B subunit of Escherichia coli labile toxin (LT-B), a 7 amino acid proline-rich linker, and 15 amino-terminal amino acids of type 5 streptococcal M protein. The purified LT-B-M5 was immunogenic in rabbits and evoked antibodies against a synthetic peptide copy of the amino-terminus of M5 (SM5[1-15]) and the native M5 protein and opsonic antibodies against type 5 streptococci. The hybrid protein retained the ganglioside-binding activity of LT-B and was tested in mice for its immunogenicity after local administration. Mice that were immunized intranasally with LT-B-M5 developed serum antibodies against SM5(1-15) and were significantly protected from death after intraperitoneal challenge infections with type 5 streptococci. The data show that protective systemic immune responses may be evoked after intranasal immunization with a fragment of M protein fused to LT-B.

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.

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.

In this study, we utilized recombinant strains expressing the M5 and M18 proteins and M- mutants of group A streptococci to compare the abilities of these M proteins to confer resistance to phagocytosis and to mediate adhesion to host cells. The data indicate that the M5 and M18 proteins can confer resistance to phagocytosis, that fibrinogen is required for this resistance, and that these M proteins can mediate adhesion to HEp-2 cells.

BACKGROUND The explanation for the very substantial decrease in the incidence of acute rheumatic fever in the United States, particularly over the past 50 years, is unclear. It has been proposed that certain M types of group A streptococci (GAS) include strains that are particularly rheumatogenic and that others are nonrheumatogenic. METHODS We compared the M type distribution of GAS recovered from children from Chicago, Illinois, with acute pharyngitis during 1961-1968 to that of GAS recovered from Chicago children and children from across the United States in 2000-2004, with attention to changes in M types that previously were associated with rheumatogenic strains. RESULTS The rheumatogenic types 3, 5, 6, 14, 18, 19, and 29 comprised 49.7% of 468 pharyngeal isolates during 1961-1968 but only 10.6% of 450 Chicago isolates during 2000-2004 (P < .001) and 17.9% of 3969 isolates nationwide during 2000-2004 (P < .001). Significant decreases in types 3, 5, and 6 and virtual disappearance of types 14, 18, 19, and 29 occurred between the 2 study periods. No change in the proportion of type 1 isolates, a highly heterogeneous group that includes some rheumatogenic strains, was observed. The nonrheumatogenic GAS types 2, 4, 22, and 28 increased from 4.9% to approximately 28% of pharyngeal isolates in Chicago and nationwide between the 2 study periods (P < .001). CONCLUSIONS These data support the concept of rheumatogenic strains of GAS and indicate that the marked decrease in the incidence of acute rheumatic fever in the United States over the past 4 decades is correlated with the replacement of rheumatogenic types by nonrheumatogenic types in cases of acute streptococcal pharyngitis in children. The reasons underlying the observed change in distribution of M types remain to be elucidated.

Background Group A streptococcus (GAS) 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 the previous obstacles to be largely overcome. Methods The vaccine is comprised of 4 recombinant proteins adsorbed to aluminum hydroxide that contain N-terminal peptides from streptococcal protective antigen and M proteins of 26 common pharyngitis, invasive, and/or rheumatogenic serotypes. Thirty healthy adult subjects received intramuscular 26-valent GAS vaccine (400 microg) at 0, 1, and 4 months, with clinical and laboratory follow-up for safety and immunogenicity using assays for tissue cross-reactive antibodies, type-specific M antibodies to 27 vaccine antigens, and functional (opsonization) activity of M protein antibodies. Results The incidence of local reactogenicity was similar to that for other aluminum hydroxide-adsorbed vaccines in adults. No subject developed evidence of rheumatogenicity or nephritogenicity, and no induction of human tissue-reactive antibodies was detected. Overall, 26 of 27 antigenic peptides evoked a >4-fold increase in the geometric mean antibody titer over baseline. The mean log2 fold-increase in serum antibody titer (+/- standard error of the mean) for all 27 antigens was 3.67 +/- 0.21. A significant mean log2 reduction in streptococcal bacterial counts in serum samples obtained after immunization was seen in opsonization assays for all M serotypes. Conclusions On the basis of epidemiological data demonstrating that the majority of cases of pharyngitis, necrotizing fasciitis, and other invasive streptococcal infections are caused by a limited number of serotypes, this 26-valent vaccine could have significant impact on the overall burden of streptococcal disease.

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes fromNeisseria meningitidisouter membrane proteins complexed with lipopolysaccharide fromShigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.

The initial step in establishing a bacterial infection is adhesion of the organism to the epithelium of the host. Group A streptococci use multiple adhesins to attach to host cells and the types of adhesins expressed by a particular strain will determine its tissue specificity. Expression of adhesins is regulated in response to changing environmental and growth conditions. Thus, the array of adhesins expressed by a group A streptococcus will depend on the complement of its adhesin genes and on the environment. Expression of some adhesins may trigger internalization of the streptococci by host cells, which may enable the streptococci to evade antibiotics and to facilitate the penetration of deeper tissues. In this review, we present the different molecular mechanisms of adhesion utilized by group A streptococci and how these interactions lead to colonization and invasion.

Virtually all group A streptococci (GAS) produce streptolysin S (SLS), a cytolytic toxin that is responsible for the beta-hemolysis surrounding colonies of the organisms grown on blood agar. SLS is an important virulence determinant of GAS, and recent studies have identified a nine-gene locus that is responsible for synthesis and transport of the toxin. SLS is not immunogenic; thus, no neutralizing antibodies are evoked during the course of natural infection. In the present study, we show that a synthetic peptide containing amino acid residues 10 to 30 of the putative SLS (SagA) propeptide [SLS(10-30)] coupled to keyhole limpet hemocyanin evoked antibodies in rabbits that completely neutralized the hemolytic activity of the toxin in vitro. Inhibition of hemolysis was reversed by preincubation of the immune serum with soluble, unconjugated peptide, indicating the specificity of the antibodies. In addition, antibodies that were affinity purified over an SLS(10-30) peptide column completely inhibited SLS-mediated hemolysis. The SLS(10-30) antisera did not opsonize group A streptococci; however, when combined with type-specific M protein antisera, the SLS antibodies significantly enhanced phagocytosis mediated by M protein antibodies. Thus, we have shown for the first time that it is possible to raise neutralizing antibodies against one of the most potent bacterial cytolytic toxins known. Our data also provide convincing evidence that the sagA gene actually encodes the SLS peptide of GAS. The synthetic peptide may prove to be an important component of vaccines designed to prevent GAS infections.

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261–1268, 1999). In this study, we have determined the complete sequence of thespagene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.

The surface M protein of group A streptococci (GAS) is one of the major virulence factors for this pathogen. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in human blood. We investigated whether pooled normal immunoglobulin G (IVIG) contains antibodies that can opsonize and enhance the phagocytosis of type M1 strains of GAS and whether the levels of these antibodies vary for different IVIG preparations. We focused on the presence of anti-M1 antibodies because the M1T1 serotype accounts for the majority of recent invasive GAS clinical isolates in our surveillance studies. The level of anti-M1 antibodies in three commercial IVIG preparations was determined by enzyme-linked immunosorbent assay (ELISA), and the opsonic activity of these antibodies was determined by neutrophil-mediated opsonophagocytosis of a representative M1T1 isolate. High levels of opsonic anti-M1 antibodies were found in all IVIG preparations tested, and there was a good correlation between ELISA titers and opsonophagocytic activity. However, there was no significant difference in the levels of opsonic anti-M1 antibodies among the various IVIG preparations or lots tested. Adsorption of IVIG with M1T1 bacteria removed the anti-M1 opsonic activity, while the level of anti-M3 opsonophagocytosis was unchanged. Plasma was obtained from seven patients with streptococcal toxic shock syndrome who received IVIG therapy, and the level of anti-M1 antibodies was assessed before and after IVIG administration. A significant increase in the level of type M1-specific antibodies was found in the plasma of all patients who received IVIG therapy ( P < 0.006). The results reveal another potential mechanism by which IVIG can ameliorate severe invasive group A streptococcal infections.

We recently concluded that M protein mediates adherence of group A streptococci to HEp-2 tissue culture cells, because the N-terminal half of M protein blocked adherence and M+ strains attached in greater numbers than M- streptococci. To further assess the role of M protein in adhesion, an M-, isogenic mutant of M type M-, isogenic mutant of M type 24 group A streptococci was constructed by insertional inactivation of the emm24 gene with the omega-interposon flanked by emm24 gene sequences. Southern blot analysis confirmed that the omega-element inserted only into emm24. The M- isogenic mutant M24-omega 3 did not react with antiserum to M24 protein, not did it survive in whole human blood. Electron micrographs of M24-omega 3 showed a diminution of surface fibrillae and reduced binding of plasma components compared with the parent strain. The adhesion of the M+ parent to HEp-2 cells and to mouse oral epithelial cells was dramatically greater than the adhesion of the M24-omega 3 mutant, although there was no difference between the two in adhesion to human buccal cells. In addition, the parent strain was dramatically more effective than the M24-omega 3 mutant in colonizing the oral cavity of mice. These results indicate that the M24 protein can serve as an adhesin in streptococcal attachment to human cells in tissue culture and is important in the colonization of mouse mucosal surfaces.

Previous studies have shown that lipoteichoic acid (LTA) of group A streptococci plays a central role in the adherence of these organisms to epithelial cells. In this study, intranasal instillation of purified LTA but not deacylated LTA in mice blocked colonization and prevented death after intranasal challenge infection with group A streptococci. Bacteria pretreated with rabbit antisera against LTA also failed to colonize or infect mice after intranasal challenge. In vitro studies showed that LTA and M protein inhibited adherence of type 24 streptococci to mouse pharyngeal cells. Passive intranasal administration of purified type 24 M protein protected mice from death after challenge infection with type 24 streptococci but had no significant effect on pharyngeal colonization. Surface LTA and M protein may mediate adherence of streptococci to mouse pharyngeal cells, and blocking adherence with LTA prevents colonization and infection in this animal model.

The roles of lipoteichoic acid (LTA) and M protein in the adherence of group A streptococci to human cells were investigated. Both M+ and M- streptococci bound to pharyngeal and buccal epithelial cells in similar numbers. Streptococcal attachment was inhibited by LTA, but not by the pepsin-extracted, amino-terminal half of M protein (pep M), suggesting that M protein does not mediate attachment to these cells. However, a purified, recombinant, intact M protein did block attachment of streptococci to buccal cells. Using synthetic peptides, the inhibitory domain was localized to a region of intact M protein that is within or near the bacterial cell wall. Evidence is presented to suggest that on the surface of streptococci this region of the M protein is probably not accessible for interactions with host cell receptors and that M protein does not mediate attachment to buccal or pharyngeal cells. In contrast, approximately 10-times more M+ streptococci bound to Hep-2 cells than did M- streptococci and pep M protein blocked binding of streptococci to Hep-2 cells. The data suggest that at least two streptococcal adhesins, LTA and M protein, are involved in the adherence of streptococci to certain cells and that the relative contributions of these adhesins to the attachment process depends on the type of host cells used to study adherence.

BackgroundHydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms.Methodology/principal findingsIsogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family.Conclusions/significanceOur findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.

Rabbit antisera evoked by type 5 streptococcal M protein (M5) were screened by enzyme-linked immunosorbent assay (ELISA) for immunological cross-reactivity with purified rabbit muscle phosphorylases a and b. Of 10 pep M5 antisera tested, 3 showed significant cross-reactivity with both forms of the enzyme. ELISA inhibition studies using one of the pep M5 antisera showed that all of the phosphorylase b antibodies were inhibited by pep M5, the immunogen, and phosphorylase b, the ELISA antigen. All of the antibodies were also inhibited by pep M6 and pep M19, but not by pep M24, indicating that the cross-reactive epitopes were shared by multiple serotypes of M protein. Western blot (immunoblot) analyses showed that pep M5 antisera reacted strongly with the subunit of phosphorylase b. In addition, purified phosphorylase partially inhibited the binding of pep M5 antibodies to a 95-kilodalton protein of human myocardium. One of the three cross-reactive pep M5 antisera inhibited the enzymatic activity of phosphorylase a in a dose-related fashion, reaching a maximum inhibition of 75%. The enzymatic activity in the presence of antibody was totally restored when the antiserum was first incubated with pep M5.

ContextGroup A streptococcal infections and their sequelae represent a global health problem. Recent advances have allowed previous obstacles associated with group A streptococcal vaccine development to be overcome.ObjectiveTo preliminarily evaluate the safety and immunogenicity of ascending doses of a recombinant fusion peptide group A streptococcal vaccine containing N-terminal M protein fragments from serotypes 1, 3, 5, 6, 19, and 24 in healthy volunteers.Design, Setting, and ParticipantsAn open-label, uncontrolled, dose-ascending phase 1 vaccine trial of 28 healthy adult volunteers aged 18 to 50 years recruited from the metropolitan area of Baltimore, Md, between October 5, 1999, and February 26, 2003, using newspaper advertisements and posted fliers, and evaluated in the outpatient facility of the Center for Vaccine Development.InterventionsEach volunteer received 3 spaced intramuscular injections of 50 µg (n = 8), 100 µg (n = 10), or 200 µg (n = 10) of hexavalent group A streptococcal vaccine formulated with aluminum hydroxide into the deltoid muscle of alternating arms.Main Outcome MeasuresAssessments of clinical safety, including elicitation of antibodies that cross-react with host tissues, and immunogenicity as measured by enzyme-linked immunosorbent assay (ELISA) and assays of opsonophagocytic- and bactericidal-antibody responses.ResultsOne year of intensive follow-up revealed the vaccine to be well tolerated. There was no evidence of tissue cross-reactive antibodies or immunological complications. At the highest (200 µg) dose, vaccination elicited significant increases in geometric mean antibody levels to all 6 component M antigens by ELISA (all P

We present evidence that a highly purified pepsin extract of type 5 streptococcal M protein (pep M5) contains at least three epitopes that are cross-reactive with sarcolemmal membrane proteins of human myocardium. The tissue-cross-reactive determinants of pep M5 are also partially shared with pep M6 and pep M19. Three rabbits immunized with a single 300 micrograms dose of pep M5 developed significant levels of heart-cross-reactive antibodies, as determined by indirect immunofluorescence tests. All three sera also contained antibodies that cross-reacted with pep M6 and pep M19. The heart tissue--specific antibodies that were eluted from sarcolemmal membranes opsonized types 5, 6, and 19 streptococci, indicating that they were directed against protective M protein epitopes on the surface of virulent organisms. Immunofluorescence inhibition tests, using purified M proteins as soluble inhibitors of heart-cross-reactive antibodies, revealed the number and M protein serotype distribution of the tissue-cross-reactive epitopes. Immunoblot analyses demonstrated the sarcolemmal membrane proteins containing the various cross-reactive antigenic determinants.

The protective immunogenicity of a hybrid peptide containing tandem copies of types 5, 6, and 24 M protein epitopes was investigated. An NH2-terminal peptide of type 24 M protein was chemically synthesized and then extended to include NH2-terminal peptides of types 6 and 5 M proteins yielding a 34-residue hybrid peptide containing a cysteine residue at its COOH-terminus. When conjugated via the cysteine residue to keyhole limpet hemocyanin (KLH), emulsified in CFA, and injected into rabbits, the synthetic hybrid evoked opsonic antibodies against types 5, 6, and 24 streptococci without stimulating tissue crossreactive immunity. The trivalent hybrid also was capable of priming T lymphocytes in vivo that responded to each of the native serotypes of M protein as well as to the synthetic hybrid peptide in vitro. The primed T cells failed to respond to the individual component peptides contained in the hybrid peptide, suggesting that the hybrid peptide confers conformations resembling the presentations of each of the subpeptides in the respective serotypes of M protein. The brisk immune responses to the type 6 peptide contained in the middle of the tandem hybrid indicates that with judicious placement between proline residues, potentially hidden peptides are readily accessible to the immune system. These results suggest that synthetic tandem peptides can be tailored in a fashion in which each of the component sets of protective epitopes can be made optimally immunoaccessible and immunogenic.