Sabrina Capo

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens

Although pili have long been recognized in Gram-negative pathogens as important virulence factors involved in adhesion and invasion, very little is known about extended surface organelles in Gram-positive pathogens. Here we report that Group A Streptococcus (GAS), a Gram-positive human-specific pathogen that causes pharyngitis, impetigo, invasive disease, necrotizing fasciitis, and autoimmune sequelae has long, surface-exposed, pilus-like structures composed of members of a family of extracellular matrix-binding proteins. We describe four variant pili and show that each is recognized by a specific serum of the Lancefield T-typing system, which has been used for over five decades to characterize GAS isolates. Furthermore, we show that immunization of mice with a combination of recombinant pilus proteins confers protection against mucosal challenge with virulent GAS bacteria. The data indicate that induction of a protective immune response against these structures may be a useful strategy for development of a vaccine against disease caused by GAS infection.

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram‐positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus‐negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three‐dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild‐type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS‐mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.

Sequence Variation in Group A Streptococcus Pili and Association of Pilus Backbone Types with Lancefield T Serotypes

BACKGROUND We previously reported that group A Streptococcus (GAS) pili are the T antigens described by Rebecca Lancefield. We also showed that these pili, constituted by backbone, ancillary 1, and ancillary 2 proteins, confer protection against GAS challenge in a mouse model. METHODS We evaluated pilus distribution and conservation by sequencing the subunits of 39 new GAS isolates and used immunoblot analysis and agglutination assays to define the specificity of T sera to pilus subunits. RESULTS GAS pili are encoded by 9 different islands within which backbone protein, ancillary protein 1, and ancillary protein 2 cluster in 15, 16, and 5 variants, respectively. Immunoblot and agglutination assays revealed that T type is determined by the backbone variant. This observation enabled us to set up a simple polymerase chain reaction assay to define the T type of GAS isolates. CONCLUSIONS We propose the use of a tee gene sequence typing, analogous to the emm gene typing, as a valuable molecular tool that could substitute for the serological T classification of GAS strains. From our sequence analysis and from recent epidemiological data, we estimate that a vaccine comprising a combination of 12 backbone variants would protect against > 90% of currently circulating strains.

Evaluation of Hepatitis C Virus Glycoprotein E2 for Vaccine Design: an Endoplasmic Reticulum-Retained Recombinant Protein Is Superior to Secreted Recombinant Protein and DNA-Based Vaccine Candidates

ABSTRACT Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Multi High-Throughput Approach for Highly Selective Identification of Vaccine Candidates: the Group A Streptococcus Case

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.

Scavenger receptor gp340 aggregates group A streptococci by binding pili.

Group A streptococci (GAS) are the most frequent cause of bacterial pharyngitis. The first obstacle to GAS colonization of the pharynx is saliva. As well as forming a physical barrier, saliva contains components of innate and acquired immunity. Previous work has shown that saliva induces bacterial aggregation, which may serve as a clearance mechanism. As the aggregation of some oral streptococci in saliva is mediated by long proteinaceous appendages, we hypothesized that pili of GAS might behave similarly. Wild‐type GAS M1 strain SF370 aggregated in saliva, while pilus‐defective mutants did not. Similarly, heterologous expression of diverse GAS pili on the surface of Lactococcus lactis induced aggregation in saliva, while control strains were unaffected. Further studies revealed that aggregating bacteria bound salivary component gp340. Purified gp340 aggregated wild‐type GAS and L. lactis expressing GAS pili, but not control strains. GAS pilus‐defective mutants were abrogated in gp340 binding and aggregation. Furthermore, gp340‐mediated aggregation reduced bacterial adhesion to human epithelial cells, suggesting a role in host defence.

Evaluation of a Group A Streptococcus synthetic oligosaccharide as vaccine candidate

Bacterial infections caused by Group A Streptococcus (GAS) are a serious health care concern that currently cannot be prevented by vaccination. The GAS cell-wall polysaccharide (GAS-PS) is an attractive vaccine candidate due to its constant expression pattern on different bacterial strains and protective properties of anti-GAS-PS antibodies. Here we report for the first time the immunoprotective efficacy of glycoconjugates with synthetic GAS oligosaccharides as compared to those containing the native GAS-PS. A series of hexa- and dodecasaccharides based on the GAS-PS structure were prepared by chemical synthesis and conjugated to CRM(197). When tested in mice, the conjugates containing the synthetic oligosaccharides conferred levels of immunoprotection comparable to those elicited by the native conjugate. Antisera from immunized rabbits promoted phagocytosis of encapsulated GAS strains. Furthermore we discuss variables that might correlate with glycoconjugate immunogenicity and demonstrate the potential of the synthetic approach that benefits from increased antigen purity and facilitated manufacturing.

Targeted Amino Acid Substitutions Impair Streptolysin O Toxicity and Group A Streptococcus Virulence

ABSTRACT Streptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin. IMPORTANCE We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site. We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.