Highly selective identification of novel vaccine candidate antigens by immunoprecipitation: the group A streptococcal case

Streptococcus pyogenes (group A streptococcus, GAS) is an obligate human pathogen that causes a spectrum of pathologies ranging from superficial infections of the skin and oropharynx to severe invasive infections of the soft tissues. Despite a steady rise in the global rate of severe GAS disease over the past 30 years, there is currently no licenced vaccine available for the prevention of GAS infection. The initial stages of GAS pathogenesis are mediated by a repertoire of proteinaceous virulence factors expressed on the bacterial cell surface that facilitate colonisation and infection through specific interactions with the host extracellular matrix. These proteins are therefore predisposed to recognition by the host immune system, which in turn makes them important targets for the development of a novel subunit vaccine. The thesis describes the development of a novel approach to the isolation and identification of the major surface antigens of GAS using twenty GAS isolates representing four serotypes actively circulating within the United Kingdom (M1, M3, M12 and M89). Antigens were purified by immunoprecipitation using an antibody formulation derived from the human clinical blood product IVIG which has been shown to protect against severe GAS infection both clinically and in vivo. This “enriched” (E-)IVIG was produced by affinity purification using a concentrated GAS cell wall protein fraction and was shown to promote neutrophil uptake in an ex vivo opsonophagocytosis assay, and impair dissemination of GAS from the nidus of infection in a murine model. A total of eight pan-serotype vaccine candidate antigens were identified by E-IVIG immunoprecipitation, seven of which were produced recombinantly for use in murine vaccination experiments. This novel approach to vaccine candidate identification could be applied to other gram positive pathogens including Staphylococcus aureus, and may have wider implications for the field of bacterial vaccinology as a whole.Open Acces

Modification of the classical Lancefield assay of group A streptococcal killing to reduce inter-donor variation

AbstractThe lack of a surrogate-of-immunity assay presents a major barrier to Streptococcus pyogenes research. Modification of the Lancefield assay to include an antibody digestion step reduced inter-donor variation and permitted detection of the anti-streptococcal activity of intravenous immunoglobulin and convalescent serum, thus facilitating retrospective evaluation of immunity using stored samples

The interaction of silver(II) complexes with biological macromolecules and antioxidants

Silver is widely used for its antimicrobial properties, but microbial resistance to heavy metals is increasing. Silver(II) compounds are more oxidizing and therefore have the potential to overcome resistance via extensive attack on cellular components, but have traditionally been hard to stabilize for biological applications. Here, the high oxidation state cation was stabilised using pyridinecarboxylate ligands, of which the 2,6-dicarboxypyridine Ag(II) complex (Ag2,6P) was found to have the best tractability. This complex was found to be more stable in phosphate buffer than DMSO, allowing studies of its interaction with water soluble antioxidants and biological macromolecules, with the aim of demonstrating its potential to oxidize them, as well as determining the reaction products. Spectrophotometric analysis showed that Ag2,6P was rapidly reduced by the antioxidants glutathione, ascorbic acid and vitamin E; the unsaturated lipids arachidonic and linoleic acids, model carbohydrate β-cyclodextrin, and protein cytochrome c also reacted readily. Analysis of the reaction with glutathione by NMR and electrospray mass spectrometry confirmed that the glutathione was oxidized to the disulfide form. Mass spectrometry also clearly showed the addition of multiple oxygen atoms to the unsaturated fatty acids, suggesting a radical mechanism, and cross-linking of linoleic acid was observed. The seven hydroxyl groups of β-cyclodextrin were found to be completely oxidized to the corresponding carboxylates. Treatment of cytochrome c with Ag2,6P led to protein aggregation and fragmentation, and dose-dependent oxidative damage was demonstrated by oxyblotting. Thus Ag2,6P was found to be highly oxidizing to a wide variety of polar and nonpolar biological molecules

Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG

SummaryObjectivesDespite over a century of research and the careful scrutiny of many promising targets, there is currently no vaccine available for the prevention of Streptococcus pyogenes infection. Through analysis of the protective, anti-streptococcal components of pooled human immunoglobulin, we previously identified ten highly conserved and invariant S. pyogenes antigens that contribute to anti-streptococcal immunity in the adult population. We sought to emulate population immunity to S. pyogenes through a process of active vaccination, using the antigens targeted by pooled human immunoglobulin.MethodsSeven targets were produced recombinantly and mixed to form a multicomponent vaccine (Spy7). Vaccinated mice were challenged with S. pyogenes isolates representing four globally relevant serotypes (M1, M3, M12 and M89) using an established model of invasive disease.ResultsVaccination with Spy7 stimulated the production of anti-streptococcal antibodies, and limited systemic dissemination of M1 and M3 S. pyogenes from an intramuscular infection focus. Vaccination additionally attenuated disease severity due to M1 S. pyogenes as evidenced by reduction in weight loss, and modulated cytokine release.ConclusionSpy7 vaccination successfully stimulated the generation of protective anti-streptococcal immunity in vivo. Identification of reactive antigens using pooled human immunoglobulin may represent a novel route to vaccine discovery for extracellular bacteria

Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis

Chemistry of the p-block elements with anionic scorpionate ligands

The chemistry of the p-block elements with both N- and S-donor anionic poly-heterocyclic borate ligands is reviewed, with an emphasis on the synthesis and structural types obtained

The bioinorganic chemistry of methimazole based soft scorpionates

Interest in the biomimetic properties of soft scorpionates based on methimazole commenced shortly after the reported synthesis of the hydrotris(methimazolyl)borate anion (TmMe) in 1996. These ligands are thione based and are thus not subject to facile oxidation and disulfide formation (c.f. thiolates). This allows these species to participate in an extensive coordination chemistry with a wide variety of metals. This review focuses on the chemistry of the RTmR and RBmR ligands with special reference to their use in biomimetic chemistry (alcohol dehydrogenase, Ada repair proteins, 5- aminolevulinate dehydratase, sulfite oxidase, hydrogenase, MerB) and their emerging use as radiopharmaceuticals (rhenium, technetium)

[Mo(TmMe)(O)2Cl]: an alternative functional model of sulfite oxidase

The hydrotris(methimazolyl)borate anion (TmMe) has been used to synthesize an alternative functional model ([Mo(TmMe)(O)2Cl]) of the metalloenzyme sulfite oxidase. It has been shown that the complex undergoes oxygen atom transfer chemistry and that it performs the primary function of the enzyme, sulfite oxidation. A method using ion chromatography has been developed to definitively prove that sulfite is oxidized to sulfate