Efficacy of Alum-Adjuvanted Peptide and Carbohydrate Conjugate Vaccine Candidates against Group A Streptococcus Pharyngeal Infection in a Non-Human Primate Model.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

Elucidating the role of N-acetylglucosamine in Group A Carbohydrate for the development of an effective glycoconjugate vaccine against Group A Streptococcus.

Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha) backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the polyRha one, both in mice and in rabbits. This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as preferable saccharide antigen to include in the vaccine.

GMMA as an Alternative Carrier for a Glycoconjugate Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM197; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM197 in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM197, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM197. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.

Immunobiology of the Classical Lancefield Group A Streptococcal Carbohydrate Antigen.

Group A Streptococcus (GAS) is a preeminent human bacterial pathogen causing hundreds of millions of infections each year worldwide. In the clinical setting, the bacterium is easily identified by a rapid antigen test against the group A carbohydrate (GAC), a polysaccharide that comprises 30 to 50% of the GAS cell wall by weight. Originally described by Rebecca Lancefield in the 1930s, GAC consists of a polyrhamnose backbone and a N-acetylglucosamine (GlcNAc) side chain. This side chain, the species-defining immunodominant antigen, is potentially implicated in autoreactive immune responses against human heart or brain tissue in poststreptococcal rheumatic fever or rheumatic heart disease. The recent discovery of the genetic locus encoding GAC biosynthesis and new insights into its chemical structure have provided novel insights into the assembly of the polysaccharide, its contribution to immune evasion and virulence, and ideas for safely harnessing its natural immunogenicity in vaccine design. This minireview serves to summarize the emerging new literature on GAC, the eponymous cell well antigen that provides structural integrity to GAS and directly interfaces with host innate and adaptive immune responses.

Immunogenicity Assessment of Cell Wall Carbohydrates of Group A Streptococcus via Self-Adjuvanted Glyco-lipopeptides.

Identifying the immunogenic moieties and their precise structure of carbohydrates plays an important role for developing effective carbohydrate-based subunit vaccines. This study assessed the structure-immunogenicity relationship of carbohydrate moieties of a single repeating unit of group A carbohydrate (GAC) present on the cell wall of group A Streptococcus (GAS) using a rationally designed self-adjuvanted lipid-core peptide, instead of a carrier protein. Immunological evaluation of fully synthetic glyco-lipopeptides (particle size: 300-500 nm) revealed that construct consisting of higher rhamnose moieties (trirhamnosyl-lipopeptide) was able to induce enhanced immunogenic activity in mice, and GlcNAc moiety was not found to be an essential component of immunogenic GAC mimicked epitope. Trirhamnosyl-lipopeptide also showed 75-97% opsonic activity against four different clinical isolates of GAS and was comparable to a subunit peptide vaccine (J8-lipopeptide) which illustrated 65-96% opsonic activity.

Rational Design of a Glycoconjugate Vaccine against Group A Streptococcus.

No commercial vaccine is yet available against Group A Streptococcus (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM197 conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM197 as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.

Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Group A Streptococcus Cell Wall Oligosaccharide-Streptococcal C5a Peptidase Conjugates as Effective Antibacterial Vaccines.

Group A streptococcus (GAS) is one of the common Gram-positive pathogenic bacteria accounting for a variety of infectious diseases. Currently, there is no commercial vaccine for GAS. To develop efficient GAS vaccines, synthetic tri-, hexa-, and nonasaccharides of a conserved group A carbohydrate (GAC) were conjugated with an inactive mutant of group A streptococcal C5a peptidase (ScpA), ScpA193, to create bivalent conjugate vaccines, which were compared with the corresponding CRM197 and TT conjugates. Systematic evaluations of these semisynthetic conjugates demonstrated that they could induce robust and comparable T-cell-dependent immune responses in mice. It was further disclosed that antibodies provoked by the ScpA193 conjugates, especially that of hexa- and nonasaccharides, could recognize and bind to GAS cells and mediate GAS opsonophagocytosis in vitro. In vivo evaluations of the hexa- and nonasaccharide-ScpA193 conjugates using a mouse model revealed that immunizing mice with especially the latter conjugate could effectively protect the animals from GAS challenges and GAS-induced pulmonary damage and significantly increase animal survival. Further in vitro studies suggested that the two ScpA193 conjugates could function through activating CD4+ T cells and promoting helper T cells (Th) to differentiate into antigen-specific Th1 and Th2 cells. In conclusion, the nonasaccharide-ScpA193 conjugate was identified as a particularly promising GAS vaccine candidate that is worthy of further investigation and development.

Group A, B, C, and G Streptococcus Lancefield antigen biosynthesis is initiated by a conserved α-d-GlcNAc-ß-1,4-l-rhamnosyltransferase.

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-ß-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter-dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-ß-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance.

Virulence Role of the GlcNAc Side Chain of the Lancefield Cell Wall Carbohydrate Antigen in Non-M1-Serotype Group A Streptococcus.

Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses.IMPORTANCE The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model.

SpyB, a Small Heme-Binding Protein, Affects the Composition of the Cell Wall in Streptococcus pyogenes.

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to ß-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.