Scilla Buccato

Identification of novel genomic islands coding for antigenic pilus‐like structures in Streptococcus agalactiae

We have recently reported the presence of covalently linked pilus‐like structures in the human pathogen, Group B Streptococcus (GBS). The pilus operon codes for three proteins which contain the conserved amino acid motif, LPXTG, associated with cell wall‐anchored proteins together with two genes coding for sortase enzymes. Analysis of the eight sequenced genomes of GBS has led to the identification of a second, related genomic island of which there are two variants, each containing genes coding for proteins with LPXTG motifs and sortases. Here we show that both variant islands also code for pilus‐like structures. Furthermore, we provide a thorough description and characterization of the genomic organization of the islands and the role of each protein in the assembly of the pili. For each pilus, polymerization of one of the three component proteins is essential for incorporation of the other two proteins into the pilus structure. In addition, two sortases are required for complete pilus assembly, each with specificity for one of the pilus components. A component protein of one of the newly identified pili is also a previously identified protective antigen and a second component of this pilus is shown to confer protection against GBS challenge. We propose that pilus‐like structures are important virulence factors and potential vaccine candidates.

Preventing Bacterial Infections with Pilus-Based Vaccines: the Group B Streptococcus Paradigm

We recently described the presence of 3 pilus variants in the human pathogen group B streptococcus (GBS; also known as Streptococcus agalactiae), each encoded by a distinct pathogenicity island, as well as the ability of pilus components to elicit protection in mice against homologous challenge. To determine whether a vaccine containing a combination of proteins from the 3 pilus types could provide broad protection, we analyzed pili distribution and conservation in 289 clinical isolates. We found that pilus sequences in each island are conserved, all strains carried at least 1 of the 3 islands, and a combination of the 3 pilus components conferred protection against all tested GBS challenge strains. These data are the first to indicate that a vaccine exclusively constituted by pilus components can be effective in preventing infections caused by GBS, and they pave the way for the use of a similar approach against other pathogenic streptococci.

Use of Lactococcus lactis Expressing Pili from Group B Streptococcus as a Broad-Coverage Vaccine against Streptococcal Disease

Recent data indicate that the human pathogen group B Streptococcus (GBS) produces pilus-like structures encoded in genomic islands with similar organization to pathogenicity islands. On the basis of the amino acid sequence of their protein components, 3 different types of pili have been identified in GBS, at least 1 of which is present in all isolates. We recently demonstrated that recombinant pilus proteins protect mice from lethal challenge with GBS and are thus potential vaccine candidates. Here, we show that GBS pilin island 1, transferred into the nonpathogenic microorganism Lactococcus lactis, leads to pilus assembly. We also show that systemically or mucosally delivered Lactococcus expressing pilin island 1 protects mice from challenge with GBS isolates carrying pilus 1. Furthermore, lactococci engineered to express hybrid pili containing GBS pilus 1 and pilus 2 components confer protection against strains expressing either of the 2 pilus types. These data pave the way to the design of pilus-based, multivalent live vaccines against streptococcal pathogens.

Rapidly produced SAM ® vaccine against H7N9 influenza is immunogenic in mice

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM® vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

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.

Environmental Acidification Drives S. pyogenes Pilus Expression and Microcolony Formation on Epithelial Cells in a FCT-Dependent Manner

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin- Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection.

The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions.

Gram‐positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT‐1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in‐frame deletion mutants, Lactococcus expressing heterologous FCT‐1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter‐bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three‐dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection.

Induction of broad-based immunity and protective efficacy by self-amplifying mRNA vaccines encoding influenza virus hemagglutinin

ABSTRACT Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, especially in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technology and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was observed for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technology could provide a rapid response platform to control pandemic influenza. IMPORTANCE In this study, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza vaccine. This novel type of vaccine elicits both humoral and cellular immune responses. Although vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make the SAM platform particularly exploitable in case of influenza pandemic.

SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.

Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens

Nucleic acid vaccines represent an attractive approach to vaccination, combining the positive attributes of both viral vectors and live-attenuated vaccines, without the inherent limitations of each technology. We have developed a novel technology, the Self-Amplifying mRNA (SAM) platform, which is based on the synthesis of self-amplifying mRNA formulated and delivered as a vaccine. SAM vaccines have been shown to stimulate robust innate and adaptive immune responses in small animals and non-human primates against a variety of viral antigens, thus representing a safe and versatile tool against viral infections. To assess whether the SAM technology could be used for a broader range of targets, we investigated the immunogenicity and efficacy of SAM vaccines expressing antigens from Group A (GAS) and Group B (GBS) Streptococci, as models of bacterial pathogens. Two prototype bacterial antigens (the double-mutated GAS Streptolysin-O (SLOdm) and the GBS pilus 2a backbone protein (BP-2a)) were successfully expressed by SAM vectors. Mice immunized with both vaccines produced significant amounts of fully functional serum antibodies. The antibody responses generated by SAM vaccines were capable of conferring consistent protection in murine models of GAS and GBS infections. Inclusion of a eukaryotic secretion signal or boosting with the recombinant protein resulted in higher specific-antibody levels and protection. Our results support the concept of using SAM vaccines as potential solution for a wide range of both viral and bacterial pathogens, due to the versatility of the manufacturing processes and the broad spectrum of elicited protective immune response.