Paolo Ruggiero

Streptococcus pneumoniae pilus subunits protect mice against lethal challenge.

ABSTRACT Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.

IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease

The lupus-like autoimmune syndrome of MRL/Mp-Tnfrsf6lpr (lpr) mice is characterized by progressive lymphadenopathy and autoantibody production, leading to early death from renal failure. Activation of T helper lymphocytes is one of the events in the pathogenesis of the disease in these mice and likely in human systemic lupus erythematosus. Among T helper lymphocytedependent cytokines, IFN-γ plays a pivotal role in the abnormal cell activation and the fatal development of the lpr disease. IL-18, an inducer of IFN-γ in T lymphocytes and natural killer cells, may contribute to the disease because cells from lpr mice are hypersensitive to IL-18 and express high levels of IL-18. To assess the contribution of IL-18 to the pathogenesis in the animal model, in vivo inhibition of IL-18 was attempted. Young lpr mice were vaccinated against autologous IL-18 by repeated administration of a cDNA coding for the murine IL-18 precursor. Vaccinated mice produced autoantibodies to murine IL-18 and exhibited a significant reduction in spontaneous lymphoproliferation and IFN-γ production as well as less glomerulonephritis and renal damage. Moreover, mortality was significantly delayed in anti-IL-18-vaccinated mice. These studies support the concept that IL-18 plays a major role in the pathogenesis of the autoimmune syndrome of lpr mice and that a reduction in IL-18 activity could be a therapeutic strategy in autoimmune diseases.

Therapeutic Vaccination against Helicobacter pylori in the Beagle Dog Experimental Model: Safety, Immunogenicity, and Efficacy

ABSTRACT Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa causing gastritis and peptic ulcer and increasing the risk of gastric cancer. The efficacy of current antibiotic-based therapies can be limited by problems of patient compliance and increasing antibiotic resistance; the vaccine approach can overcome these limits. The present study describes the therapeutic vaccination of experimentally H. pylori-infected beagle dogs, an animal model that reproduces several aspects of the human infection with H. pylori. The vaccine consisted of three recombinant H. pylori antigens, CagA, VacA, and NAP, formulated at different doses (10, 25, or 50 μg each) with alum and administered intramuscularly either weekly or monthly. No adverse effects were observed after vaccination and a good immunoglobulin G response was generated against each of the three antigens. Bacterial colonization and gastritis were decreased after the completion of the vaccination cycle, especially in the case of the monthly immunization schedule. In conclusion, therapeutic vaccination in the beagle dog model was safe and immunogenic and was able to limit H. pylori colonization and the related gastric pathology.

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.

The quest for a vaccine against Helicobacter pylori: how to move from mouse to man?

Several lines of evidence from experimental animal models of infection have clearly demonstrated the feasibility of a prophylactic and therapeutic vaccine against Helicobacter pylori. However, comparatively few clinical studies have been carried out to evaluate whether the positive results obtained in animals can be reproduced in humans. The preliminary results obtained with single component, mucosally delivered vaccines have shown very limited results thus far. Very good immunogenicity and safety profiles are now being obtained with parenterally delivered, aluminium hydroxide-adjuvanted multicomponent candidate vaccines. For sure, better vaccine formulations, better antigen preparation(s), better adjuvants, and better delivery systems have to be designed and tested for safety and immunogenicity. These studies are also needed for deciphering those aspects of the effector immune responses that correlate with protection against H. pylori infection and disease.

Plant polyphenols inhibit VacA, a toxin secreted by the gastric pathogen Helicobacter pylori

VacA is a major virulence factor of the widespread stomach‐dwelling bacterium Helicobacter pylori. It causes cell vacuolation and tissue damage by forming anion‐selective, urea‐permeable channels in plasma and endosomal membranes. We report that several flavone derivatives and other polyphenols present in vegetables and plants inhibit ion and urea conduction and cell vacuolation by VacA. Red wine and green tea, which contain many of the compounds in question, also potently inhibit the toxin. These observations suggest that polyphenols or polyphenol derivatives may be useful in the prevention or cure of H. pylori‐associated gastric diseases.

Enhanced mucosal and systemic immune responses to Helicobacter pylori antigens through mucosal priming followed by systemic boosting immunizations

It is estimated that Helicobacter pylori infects the stomachs of over 50% of the worlds population and if not treated may cause chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B‐cell lymphoma. The aim of this study was to enhance the mucosal and systemic immune responses against the H. pylori antigens cytotoxin‐associated gene A (CagA) and neutrophil‐activating protein (NAP), through combinations of mucosal and systemic immunizations in female BALB/c mice. We found that oral or intranasal (i.n.) followed by i.m. immunizations induced significantly higher serum titres against NAP and CagA compared to i.n. alone, oral alone, i.m. alone, i.m. followed by i.n. or i.m. followed by oral immunizations. However, only oral followed by i.m. immunizations induced anti‐NAP antibody‐secreting cells in the stomach. Moreover, mucosal immunizations alone or in combination with i.m., but not i.m. immunizations alone, induced mucosal immunoglobulin A (IgA) responses in faeces. Any single route or combination of immunization routes with NAP and CagA preferentially induced antigen‐specific splenic interleukin‐4‐secreting cells and far fewer interferon‐γ‐secreting cells in the spleen. Moreover, i.n. immunizations alone or in combination with i.m. immunizations induced predominantly serum IgG1 and far less serum IgG2a. Importantly, we found that while both i.n. and i.m. recall immunizations induced similar levels of serum antibody responses, mucosal IgA responses in faeces were only achieved through i.n. recall immunization. Collectively, our data show that mucosal followed by systemic immunization significantly enhanced local and systemic immune responses and that i.n. recall immunization is required to induce both mucosal and systemic memory type responses.

Structure-function relationship in the IL-1 family.

The interleukin 1 (IL-1) family is a group of related cytokines including two agonist proteins (IL-1alpha and IL-1beta), each derived by enzymatic cleavage of precursor proteins (pro-IL-1alpha and pro-IL-1beta), and three forms of an antagonist protein (IL-1ra, icIL-1raI, icIL-1raII). IL-1 plays a key role in the onset and development of the host reaction to invasion, being an important factor in the initiation of the inflammatory response and in the triggering of immune functions. Due to its pleiotropic activity and to the high potency of its inflammatory effects, IL-1 activity is tightly regulated in the body by a complex network of control systems. These include the presence of two types of inhibitors, the receptor antagonist IL-1ra and the second type of IL-1 receptor (IL-1RI), which is a natural scavenger of IL-1. Furthermore, regulation of IL-1 activity is attained by a strict hierarchy of binding affinity of the two receptors (the activating IL-1RI and the inhibitory IL-1RII) for the various members of the IL-1 family. Additional levels of control are represented by the presence of soluble forms of both receptors and of immature pro-IL-1 forms with different characteristics of activity and receptor binding capacity. To clarify the features of reciprocal interaction among ligands and receptors, in the attempt to understand the rules regulating the IL-1 system and its effectiveness, a deep analysis of the relationship between structure and function in the proteins of the IL-1 family becomes of key importance. Information on this line has been provided by several groups mainly with studies of mutagenesis of IL-1alpha, IL-1beta and IL-1ra in parallel with biological assays of activity. In this review, a survey of the available data is provided, in order to construct a hypothetical model of the functional structure of IL-1 proteins as a basis for future therapeutic interventions based on genetic and protein engineering.

The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic calcium oscillations.

Helicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro‐inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro‐inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL‐2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium‐dependent production of pro‐inflammatory cytokines, including tumour necrosis factor α (TNF‐α). These observations indicate that VacA may act as a pro‐inflammatory factor of H. pylori at very early stages of the innate immune response.

Helicobacter pylori CagA: From Pathogenic Mechanisms to Its Use as an Anti-Cancer Vaccine

Helicobacter pylori colonizes the gastric mucosa of more than 50% of the human population, causing chronic inflammation, which however is largely asymptomatic. Nevertheless, H. pylori-infected subjects can develop chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Chronic exposure to the pathogen and its ability to induce epithelial to mesenchymal transition (EMT) through the injection of cytotoxin-associated gene A into gastric epithelial cells may be key triggers of carcinogenesis. By deregulating cell–cell and cell–matrix interactions as well as DNA methylation, histone modifications, expression of micro RNAs, and resistance to apoptosis, EMT can actively contribute to early stages of the cancer formation. Host response to the infection significantly contributes to disease development and the concomitance of particular genotypes of both pathogen and host may turn into the most severe outcomes. T regulatory cells (Treg) have been recently demonstrated to play an important role in H. pylori-related disease development and at the same time the Treg-induced tolerance has been proposed as a possible mechanism that leads to less severe disease. Efficacy of antibiotic therapies of H. pylori infection has significantly dropped. Unfortunately, no vaccine against H. pylori is currently licensed, and protective immunity mechanisms against H. pylori are only partially understood. In spite of promising results obtained in animal models of infection with a number of vaccine candidates, few clinical trials have been conducted so far and with no satisfactory outcomes. However, prophylactic vaccination may be the only means to efficiently prevent H. pylori-associated cancers.