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MARCOIL heptad position assignment of the N-terminal regions of M1, M238, M227, and M239 projected onto a helical wheel generated using DRAWCOIL 1.0. Only heptad repeat regions with probability percentages of 2% were considered for helical wheel representation. The view is from the N-terminal region to the C-terminal region. Heptad repeat positions are labeled a-g. The following color scheme is followed: polar positive, blue; polar negative, red; polar neutral, orange; and nonpolar aliphatic, gray.
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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 consider...
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... (MAR-COIL (26) assigned coiled-coil probability 2%) and calculated the sequence identity between each of the corresponding heptad sites of vaccine and nonvaccine M types. The heptad repeat projected on a helical wheel generated using DRAWCOIL 1.0 (27) for M types that share high overall sequence identity with vaccine type M1 (40%) is shown in Fig. ...
Citations
... Therefore, an alternative explanation for simultaneous antibody reactivity to both CT1230N and CT1230C antigens in almost half of the serum samples (77 of 157 samples) reactive to either of these antigens could probably stem from cross-reactive epitopes in these antigens. Although coiled-coil heptad repeat sequence identity could contribute to cross-reactive epitopes, it seems that amino acid identity/similarity at solvent-exposed positions in heptad repeats may influence antibody cross-reactivity, an issue responsible for antibody cross-reactivity among several variants of streptococcal M proteins 33 . Recently, a number of cross-reactive antibodies recognizing α-helical coiled-coil peptides derived from the orthologous sexual stage antigens of P. falciparum and P. vivax have been reported 34 . ...
Merozoite surface protein 3 of Plasmodium vivax (PvMSP3) contains a repertoire of protein members with unique sequence organization. While the biological functions of these proteins await elucidation, PvMSP3 has been suggested to be potential vaccine targets. To date, studies on natural immune responses to this protein family have been confined to two members, PvMSP3α and PvMSP3β. This study analyzed natural IgG antibody responses to PvMSP3γ recombinant proteins derived from two variants: one containing insert blocks (CT1230nF) and the other without insert domain (NR25nF). The former variant was also expressed as two subfragment proteins: one encompassing variable domain I and insert block A (CT1230N) and the other spanning from insert block B to conserved block III (CT1230C). Serum samples were obtained from 246 symptomatic vivax malaria patients in Tak (n = 50) and Ubon Ratchathani (n = 196) Provinces. In total, 176 (71.5%) patients could mount antibodies to at least one recombinant PvMSP3γ antigen. IgG antibodies directed against antigens CT1230nF, CT1230N, CT1230C and NR25nF occurred in 96.6%, 61.4%, 71.6% and 68.2% of samples, respectively, suggesting the widespread occurrence of B-cell epitopes across PvMSP3γ. The rates of seropositivity seemed to correlate with the number of previous malaria episodes. Isotype analysis of anti-PvMSP3γ antibodies has shown predominant cytophilic subclass responses, accounting for 75.4–81.7% for IgG1 and 63.6–77.5% for IgG3. Comparing with previous studies in the same cohort, the numbers of serum samples reactive to antigens derived from P. vivax merozoite surface protein 9 (PvMSP9) and thrombospondin-related anonymous protein (PvTRAP) were higher than those to PvMSP3γ, being 92.7% and 87.0% versus 71.5%, respectively. Three (1.22%) serum samples were nonresponsive to all these malarial proteins. Nevertheless, the relevance of naturally acquired antibodies to PvMSP3γ in host protection requires further studies.
... In fact, crossreactivity has become one of the main hypothesized mechanisms causing the emergence of ARF attacks several weeks after the onset of manifestations of GAS infection such as sore throat infection or impetigo due to autoimmunity which recognizes the M-protein GAS along with the proteins found in the heart valves, synovial tissue, and basal ganglia due to the similarity of protein structures which is referred to as molecular mimicry. 8, [74][75][76][77] The growing interest in developing GAS vaccines based on non-M protein antigens stems from a desire to avoid GAS vaccine targets that may cause autoimmunity. The development of non-M protein vaccines began in the 1990s using C5a peptidase (Streptococcal C5a protease, SCPA). ...
Group A Streptococcus (GAS) is a widely distributed bacterium that is Gram-positive and serves as the primary cause of acute rheumatic fever (ARF) episodes. Rheumatic heart disease (RHD) is a sequela resulting from repeated ARF attacks which are also caused by repeated GAS infections. ARF/RHD morbidity and mortality rates are incredibly high in low- and middle-income countries. This is closely related to poor levels of sanitation which causes the enhanced incidence of GAS infections. Management of carditis in RHD cases is quite challenging, particularly in developing countries, considering that medical treatment is only palliative, while definitive treatment often requires more invasive procedures with the high costs. Preventive action through vaccination against GAS infection is one of the most effective steps as a solution in reducing RHD morbidity and mortality due to curative treatments are expensive. Various developments of M-protein-based GAS vaccines have been carried out over the last few decades and have recently begun to enter the clinical stage. Nevertheless, this vaccination generates cross-reactive antibodies that might trigger ARF assaults as a result of the resemblance between the M-protein structure and proteins found in many human tissues. Consequently, the development of a vaccine utilizing L-Rhamnose derived from the poly-rhamnose backbone of Group A Carbohydrate (GAC) commenced. The L-Rhamnose-based vaccine was chosen due to the absence of the Rhamnose biosynthesis pathway in mammalian cells including humans thus this molecule is not found in any body tissue. Recent pre-clinical studies reveal that L-Rhamnose-based vaccines provide a protective effect by increasing IgG antibody titers without causing cross-reactive antibodies in test animal tissue. These findings demonstrate that the L-Rhamnose-based vaccine possesses strong immunogenicity, which effectively protects against GAS infection while maintaining a significantly higher degree of safety.
... The M protein is an α-helical dimer located on the GAS surface. The basic structural components of the M protein consist of an N-terminal hypervariable region, central domain, and C-terminal conserved region [14]. Due to the high variability of the N terminus of the M protein, it exhibits quite complex antigenic diversity. ...
Streptococcus pyogenes (group A Streptococcus; GAS), a Gram-positive coccal bacterium, poses a significant global disease burden, especially in low- and middle-income countries. Its manifestations can range from pharyngitis and skin infection to severe and aggressive diseases, such as necrotizing fasciitis and streptococcal toxic shock syndrome. At present, although GAS is still sensitive to penicillin, there are cases of treatment failure for GAS pharyngitis, and antibiotic therapy does not universally prevent subsequent disease. In addition to strengthening global molecular epidemiological surveillance and monitoring of antibiotic resistance, developing a safe and effective licensed vaccine against GAS would be the most effective way to broadly address GAS-related diseases. Over the past decades, the development of GAS vaccines has been stalled, mainly because of the wide genetic heterogeneity of GAS and the diverse autoimmune responses to GAS. With outbreaks of scarlet fever in various countries in recent years, accelerating the development of a safe and effective vaccine remains a high priority. When developing a GAS vaccine, many factors need to be considered, including the selection of antigen epitopes, avoidance of self-response, and vaccine coverage. Given the challenges in GAS vaccine development, this review describes the important virulence factors that induce disease by GAS infection and how this has influenced the progression of vaccine development efforts, focusing on several candidate vaccines that are further along in development.
... However, with the advent of emm sequencing, it has become evident that the majority of M types are structurally related and can be grouped into sequence-based clusters that share host protein binding characteristics [12]. This observation and our previous results showing that a 30-valent vaccine elicited antibodies that cross-opsonized multiple "non-vaccine" M types [6] prompted us to take a structure-based approach to the design of multivalent M peptide vaccines [13][14][15]. ...
... Therefore, similarity in epitope 3D structure between two sequence-related M peptides is hypothesized to predict antibody crossreactivity. Here, we used a set of structural criteria shown to predict with high specificity the likelihood of eliciting significant levels of crossreactive antibodies between pairs of sequence-related heterologous M peptides [13][14][15]. Previous studies have identified 117 sequence-related N-terminal M peptides from M types that are responsible for >90 % of Strep A infections globally [14]. ...
... Here, we used a set of structural criteria shown to predict with high specificity the likelihood of eliciting significant levels of crossreactive antibodies between pairs of sequence-related heterologous M peptides [13][14][15]. Previous studies have identified 117 sequence-related N-terminal M peptides from M types that are responsible for >90 % of Strep A infections globally [14]. After assessing pairwise sequence identity of all 117 peptides, additional criteria considered the empirical heptad identity scores of the peptide pairs, the length of the predicted coiled-coil region and the degree of coiled-coil propensity. ...
The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal region of the M protein is variable in sequence, defines the M/emm type, and contains epitopes that elicit opsonic antibodies that protect animals from challenge infections. Although there are >200 M types of Strep A, there is now evidence that structurally related M proteins can be grouped into clusters and that immunity may be cluster-specific in addition to M type-specific. This observation has led to recent studies of structure-based design of multivalent M peptide vaccines to select peptides predicted to cross-react with heterologous M types to improve vaccine coverage. In the current study, we have applied a refined series of peptide structural algorithms to predict immunological cross-reactivity among 117 N-terminal M peptides representing the most prevalent M types of Strep A. Based on the results of the structural analyses, in combination with global M type prevalence data, we constructed a 32-valent vaccine containing 19 cross-reactive vaccine candidates predicted to cross-react with 37 heterologous M peptides to which were added 13 type-specific M peptides. The 4-protein recombinant vaccine was immunogenic in rabbits and elicited significant levels of antibodies against 31/32 (97%) vaccine peptides and 28/37 (76%) peptides predicted to cross-react. The vaccine antisera also promoted opsonophagocytic killing of vaccine and cross-reactive M types of Strep A. Based on a recent analysis of M type prevalence of Strep A, the potential global coverage of the 32-valent vaccine is ∼90%, ranging from 68% in Africa to 95% in North America. Our results indicate the utility of structure-based design that may be applied to future studies of broadly protective M peptide vaccines.
... This region contains epitopes that elicit Abs with the greatest opsonic (protective) potential (21,22) and are least likely to elicit potentially harmful Abs that may crossreact with human tissues (10,12,23,24). We previously constructed a phylogenetic sequence-based tree of 117 N-terminal (50 aa) M peptides (17) from epidemiologically important M types of Strep A and divided them into seven sequence-related clusters (N-terminal clusters [NTC]) based on loosely rooted branches (Fig. 1C). In a recent study, we focused on one sequence-based cluster containing 21 M types (NTC6), using a combination of sequence identity, Ab binding, and cheminformatics to select six vaccine peptides that were predicted to cross-react with all 15 nonvaccine M peptides (17). ...
... We previously constructed a phylogenetic sequence-based tree of 117 N-terminal (50 aa) M peptides (17) from epidemiologically important M types of Strep A and divided them into seven sequence-related clusters (N-terminal clusters [NTC]) based on loosely rooted branches (Fig. 1C). In a recent study, we focused on one sequence-based cluster containing 21 M types (NTC6), using a combination of sequence identity, Ab binding, and cheminformatics to select six vaccine peptides that were predicted to cross-react with all 15 nonvaccine M peptides (17). The vaccine antisera cross-reacted with 10 of the 15 nonvaccine peptides. ...
... Retrospective structural analysis revealed that significant sequence identity at corresponding polar amino acid sites within the coiled-coil a-helical heptad repeats between vaccine and nonvaccine peptides accurately distinguished cross-reactive from noncrossreactive peptides. We subsequently developed a scoring algorithm (17) based on the sequence identity at polar heptad sites. In the current study, we improve significantly upon the previous algorithm. ...
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.
... We have previously shown that the sequence and structural similarities within M clusters can be exploited to formulate vaccines containing a minimum number of N-terminal M peptides predicted to cross-react with heterologous M types in the same cluster [12,15]. In these studies, the predictive algorithms included sequence identity, three-dimensional conformation, and chemin formatics of the 1-50 aa or 16-50 aa N-terminal M peptides. ...
... To maximize the selection of cross-reactive epitopes, ABCpred linear epitopes were submitted to the IEDB epitope conservancy analysis tool, which searches for sequences similar to user-defined epitope sequences within a set of antigens [20]. A sequence identity threshold of 60% was chosen to identify potential cross-reactive epitopes conserved among heterologous M types, based on previous observations of cross-reactive immune responses among sequence-based clusters of N-terminal M peptides [12,15]. ...
... Our initial assessment was comprehensive and designed to identify all potential crossreactive epitopes present in N-terminal M sequences from 117 epidemiologically significant [15] Strep A M types. These 117 M types account for approximately 88% of all Strep A infections globally [25]. ...
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.
... With this in mind, Collado et al. [92] and Perrig and colleagues [93], considered the possible use of additional Staphylococcus peptides, such as S. uberis adhesion molecule (SUAM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase (FBA), elongation factor Ts (EFTs), mtuA, and an unspecified fibronectin-binding protein. In addition, several studies aimed to develop vaccines against a variety of Streptococcus group A proteins, including the M protein, pili components, adhesins, and the C5a protease [94][95][96][97][98][99]. ...
Streptococcus spp. are major mastitis pathogens present in dairy products, which produce a variety of virulence factors that are involved in streptococcal pathogenicity. These include neuraminidase, pyrogenic exotoxin, and M protein, and in addition they might produce bacteriocins and antibiotic-resistance proteins. Unjustifiable misuse of antimicrobials has led to an increase in antibiotic-resistant bacteria present in foodstuffs. Identification of the mastitis-causing bacterial strain, as well as determining its antibiotic resistance and sensitivity is crucial for effective therapy. The present work focused on the LC–ESI–MS/MS (liquid chromatography–electrospray ionization tandem mass spectrometry) analysis of tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2706 non-redundant peptides belonging to 2510 proteins was identified and analyzed. Among them, 168 peptides were determined, representing proteins that act as virulence factors, toxins, anti-toxins, provide resistance to antibiotics that are associated with the production of lantibiotic-related compounds, or play a role in the resistance to toxic substances. Protein comparisons with the NCBI database allowed the identification of 134 peptides as specific to Streptococcus spp., while two peptides (EATGNQNISPNLTISNAQLNLEDKNK and DLWC*NM*IIAAK) were found to be species-specific to Streptococcus dysgalactiae. This proteomic repository might be useful for further studies and research work, as well as for the development of new therapeutics for the mastitis-causing Streptococcus strains.
Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (Strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220 M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens due to type-specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was recently shown to elicit M type cross-reactivity. The basis for this cross-reactivity is unknown, but may be due in part to antibody recognition of a three-dimensional (3D) pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit cross-reactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S. pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum recognized M proteins displayed natively on the Strep A surface and promoted the opsonophagocytic killing of Strep A strains expressing these M proteins. As C4BP-binding is a conserved virulence trait of Strep A, we propose targeting the 3D pattern may prove advantageous in vaccine design.
