Luigi Fiaschi

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Significance Staphylococcus aureus is a human pathogen causing life-threatening infections. The high incidence of methicillin-resistant S. aureus isolates resistant to all antibiotics makes the development of anti-S. aureus vaccines an urgent medical need. However, the unique ability of S. aureus to produce virulent factors, which counteract virtually all pathways of innate and adaptive immunity, has hampered all vaccine discovery efforts. Starting from the assumption that to be effective a vaccine should induce highly functional antibodies and potentiate the killing capacity of phagocytic cells, we selected a cocktail of five conserved antigens involved in different mechanisms of pathogenesis, and we formulated them with a potent adjuvant. This vaccine provides an unprecedented protective efficacy against S. aureus infection in animal models. Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7–10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17–secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

Designing the Next Generation of Vaccines for Global Public Health

Vaccine research and development are experiencing a renaissance of interest from the global scientific community. There are four major reasons for this: (1) the lack of efficacious treatment for many devastating infections; (2) the emergence of multidrug resistant bacteria; (3) the need for improving the safety of the more traditional licensed vaccines; and finally, (4) the great promise for innovative vaccine design and research with convergence of omics sciences, such as genomics, proteomics, immunomics, and vaccinology. Our first project based on omics was initiated in 2000 and was termed reverse vaccinology. At that time, antigen identification was mainly based on bioinformatic analysis of a singular genome. Since then, omics-guided approaches have been applied to its full potential in several proof-of-concept studies in the industry, with the first reverse vaccinology-derived vaccine now in late stage clinical trials and several vaccines developed by omics in preclinical studies. In the meantime, vaccine discovery and development has been further improved with the support of proteomics, functional genomics, comparative genomics, structural biology, and most recently vaccinomics. We illustrate in this review how omics biotechnologies and integrative biology are expected to accelerate the identification of vaccine candidates against difficult pathogens for which traditional vaccine development has thus far been failing, and how research will provide safer vaccines and improved formulations for immunocompromised patients in the near future. Finally, we present a discussion to situate omics-guided rational vaccine design in the broader context of global public health and how it can benefit citizens in both developed and developing countries.

Staphylococcus aureus FhuD2 Is Involved in the Early Phase of Staphylococcal Dissemination and Generates Protective Immunity in Mice

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target.

Protective Efficacy Induced by Recombinant Clostridium difficile Toxin Fragments

ABSTRACT Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.

Diversity of cwp loci in clinical isolates of Clostridium difficile.

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.

Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

ABSTRACT Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.