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

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.

CagL from Helicobacter pylori has ADP-ribosylation activity and exerts partial protective efficacy in mice.

Mono ADP-ribosyltransferases are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In prokaryotes, mono ADP-ribose transfer enzymes often represent a family of exotoxins that display activity in a variety of bacteria responsible for causing disease in plants and animals. A bioinformatic approach has allowed us to identify that CagL gene from some Helicobacter pylori strains shares a sequence pattern with ADP-ribosylating toxins of the CT-group. In this manuscript we show that recombinant CagL from Shi470 is catalytically active showing ADP-ribosyltransferase, NAD-glycohydrolase, and auto-ADP-ribosylation activities. This is the first time that a catalytically active member of the ADP-ribosyltransferase family is identified in Helicobacter pylori. This observation may lead to the discovery of novel functions exerted by CagL in the pathogenesis of Helicobacter pylori. Indeed, we have shown that vaccination with CagL has protective efficacy in mice indicating that CagL may be considered as potential component of a Helicobacter pylori vaccine.

Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.

Exogenous sialic acid transport contributes to group B streptococcus infection of mucosal surfaces.

By sequence analysis of available group B streptococcus (GBS) genomes, we discovered a conserved putative operon involved in the catabolism of sialic acid, containing a tripartite transporter formed by two integral membrane components and a sugar-binding unit, named SAL0039. Expression analysis in the presence of different substrates revealed that SAL0039 was specifically upregulated by the presence of sialic acid and downregulated when bacteria were grown in human blood or in the presence of a high concentration of glucose. The role of SAL0039 in sugar transport was supported by the inability of the sal0039 deletion mutant strain to import exogenous sialic acid and to grow in semidefined medium supplemented with this sugar. Furthermore, in vivo evidence showed that the presence of exogenous sialic acid significantly increased the capacity of GBS to infect mice at the mucosal level. These findings suggest that transport of sialic acid may also contribute to GBS infections.

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain.

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.

Helicobacter pylori infection: what's new.

PURPOSE OF REVIEW: This review is aimed at describing the main findings of 2011 on the aspects of Helicobacter pylori-related gastric disease linked to CagA and to T-regulatory cells (Treg), and on the attempts to improve the treatment efficacy. RECENT FINDINGS: Recent findings presented in this review are as follows: CagA interferes with tumor suppression; tolerance protects from H. pylori-induced disease; modified H. pylori treatments/regimens can afford higher efficacy than the standard triple therapy. SUMMARY: H. pylori colonizes the human stomach causing gastritis and severe diseases including gastric cancer. One of the most dangerous H. pylori factors, CagA, has been investigated in relation to gastric cancer: recently this relationship was strongly reinforced by the finding that CagA interacts with the tumor suppressor apoptosis-stimulating protein of p53-2 (ASPP2), promoting p53 degradation. Treg have been proposed to be involved in H. pylori infection and gastric disease: recent findings suggest that Treg-induced tolerance, rather than immunity to H. pylori, may result in less severe disease. The eradication rates achieved with the standard triple therapy dropped below 80%, mainly due to antibiotic resistance, while no vaccines are currently licensed; new treatments/regimens were subjected to clinical trials, in some cases strongly increasing the eradication rates.

The two variants of the Streptococcus pneumoniae pilus 1 RrgA adhesin retain the same function and elicit cross-protection in vivo.

Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the "head" of the protein, which contains the putative binding domains, whereas the elongated "stalk" was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the "head" domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Cholera toxin and Escherichia coli heat-labile enterotoxin, but not their nontoxic counterparts, improve the antigen-presenting cell function of human B lymphocytes.

B lymphocytes play an important role in the immune response induced by mucosal adjuvants. In this study we investigated the in vitro antigen-presenting cell (APC) properties of human B cells upon treatment with cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) and nontoxic counterparts of these toxins, such as the B subunit of CT (CT-B) and the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Furthermore, forskolin (FSK), a direct activator of adenylate cyclase, and cyclic AMP (cAMP) analogues were used to investigate the role of the increase in intracellular cAMP caused by the A subunit of CT and LT. B lymphocytes were cultured with adjuvants and polyclonal stimuli necessary for activation of B cells in the absence of CD4 T cells. Data indicated that treatment with CT, LT, FSK, or cAMP analogues, but not treatment with CT-B or LTK63, upregulated surface activation markers on B cells, such as CD86 and HLA-DR, and induced inhibition of the proliferation of B cells at early time points, while it increased cell death in long-term cultures. Importantly, B cells treated with CT, LT, or FSK were able to induce pronounced proliferation of both CD4(+) and CD8(+) allogeneic T cells compared with untreated B cells and B cells treated with CT-B and LTK63. Finally, only treatment with toxins or FSK induced antigen-specific T-cell proliferation in Mycobacterium tuberculosis purified protein derivative or tetanus toxoid responder donors. Taken together, these results indicated that the in vitro effects of CT and LT on human B cells are mediated by cAMP.

Sortase A confers protection against Streptococcus pneumoniae in mice.

Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.

Protective effect of Group B Streptococcus type-III polysaccharide conjugates against maternal colonization, ascending infection and neonatal transmission in rodent models.

Group B Streptococcus (GBS) is a normal inhabitant of recto-vaginal mucosae in up to 30% of healthy women. Colonization is a major risk factor for perinatal infection which can lead to severe complications such as stillbirth and neonatal invasive disease. Intra-partum antibiotic prophylaxis in colonized women is a safe and cost-effective preventive measure against early-onset disease in the first days of life, but has no effect on late-onset manifestations or on early maternal infection. Maternal immunization with capsular polysaccharide-based vaccines shows promise for the prevention of both early-onset and late-onset neonatal infections, although ability to prevent maternal colonization and ascending infection has been less studied. Here we investigated the effect of a GBS glycoconjugate vaccine since the very early stage of maternal GBS acquisition to neonatal outcome by rodent models of vaginal colonization and ascending infection. Immunization of female mice and rats with a type III glycoconjugate reduced vaginal colonization, infection of chorioamniotic/ placental membranes and bacterial transmission to fetuses and pups. Type III specific antibodies were detected in the blood and vagina of vaccinated mothers and their offspring. The obtained data support a potential preventive effect of GBS glycoconjugate vaccines during the different stages of pregnancy.

Efficacy, immunogenicity, and safety of a parenteral vaccine against Helicobacter pylori in healthy volunteers challenged with a Cag-positive strain: a randomised, placebo-controlled phase 1/2 study.

BACKGROUND: Intramuscular immunisation with a vaccine composed of three recombinant Helicobacter pylori antigens-vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP)-prevented infection in animal models and was well tolerated and highly immunogenic in healthy adults. We aimed to assess the efficacy of the vaccine in prevention of a H pylori infection after challenge with a CagA-positive strain (BCM 300) in healthy volunteers. METHODS: In this randomised phase 1/2, observer-blind, placebo-controlled, single-centre study, healthy non-pregnant adults aged 18-40 years who were confirmed negative for H pylori infection were randomly assigned (3:4) to three intramuscular doses of either placebo or vaccine at 0, 1, and 2 months. Randomisation was via a computer-generated list with study numbers ensuring the correct ratio within a block size of seven. Participants were consecutively assigned in a double-blind manner to existing study numbers of the study protocol. Investigators and participants were blinded to allocation throughout the study. One month after the third immunisation, participants underwent challenge with a CagA-positive H pylori strain, which, for safety reasons, was initially administered in a subset of participants. The primary efficacy outcome was the efficacy of the vaccine as measured by the proportion of participants infected with H pylori 12 weeks after the challenge. At the end of the study, participants infected with H pylori were treated for 14 days with combination therapy consisting of a proton pump inhibitor and two antibiotics twice daily. Safety and immunogenicity were monitored at pre-established visits. This trial is registered with ClinicalTrials.gov, number NCT00736476, and is completed. FINDINGS: 63 patients were randomly assigned, 27 to placebo and 36 to the vaccine. 34 participants (19 in the vaccinated group and 15 in the placebo group) underwent infectious challenge, all but one of whom experienced transient mild-to-moderate epigastric symptoms. 12 weeks after infectious challenge, six (32%) of 19 people in the vaccinated group and six (40%) of 15 people in the placebo group remained positive for H pylori. Eradication was successful in everyone who remained infected at 12 weeks. The geometric mean concentrations of antibodies specific to CagA (202 [95% CI 69-588] vs 4·73 [95% CI 1·41-16]; p=0·001), VacA (1469 [838-2577] vs 73 [39-138]; p=0·001), and NAP (208 [139-313] vs 8·01 [5·05-13]; p=0·001) were significantly higher in the vaccine group than in the placebo group 12 weeks after infectious challenge. INTERPRETATION: Compared with placebo, the vaccine did not confer additional protection against H pylori infection after challenge with a CagA-positive strain, despite increased systemic humoral responses to key H pylori antigens. The finding of spontaneous clearance of H pylori infection in more than half the participants in the placebo group is remarkable and suggests important immune protection in the healthy adult population. FUNDING: Novartis Vaccine and Diagnostics.

Red wine and green tea reduce H pylori- or VacA-induced gastritis in a mouse model.

AIM: To investigate whether red wine and green tea could exert anti-H pylori or anti-VacA activity in vivo in a mouse model of experimental infection. METHODS: Ethanol-free red wine and green tea concentrates were administered orally as a mixture of the two beverages to H pylori infected mice, or separately to VacA-treated mice. Gastric colonization and gastric inflammation were quantified by microbiological, histopathological, and immunohistochemical analyses. RESULTS: In H pylori-infected mice, the red wine and green tea mixture significantly prevented gastritis and limited the localization of bacteria and VacA to the surface of the gastric epithelium. Similarly, both beverages significantly prevented gastric epithelium damage in VacA-treated mice; green tea, but not red wine, also altered the VacA localization in the gastric epithelium. CONCLUSION: Red wine and green tea are able to prevent H pylori-induced gastric epithelium damage, possibly involving VacA inhibition. This observation supports the possible relevance of diet on the pathological outcome of H pylori infection.

Prevalence of M75 Streptococcus pyogenes Strains Harboring slaA Gene in Patients Affected by Pediatric Obstructive Sleep Apnea Syndrome in Central Italy.

Recently we reported an association between pediatric obstructive sleep apnea syndrome (OSAS) and Group A streptococcus (GAS) sub-acute chronic tonsil colonization. We showed that GAS may contribute to tonsil hyperplasia via a streptolysin O (SLO)-dependent cysteinyl leukotrienes (CysLTs) production, which can trigger T and B cell proliferation. In the present study, we characterized the GAS strains isolated from pediatric OSAS patients in comparison with a panel of age and sex matched GAS strains unrelated to OSAS, but isolated in the same area and during the same period ranging from 2009 to 2013. We found that slaA gene, previously reported to be associated to CysLTs production pathway, was significantly associated to GAS OSAS strains. Moreover, the most numerous group (32%) of the GAS OSAS strains belonged to M75 type, and 6 out of 7 of these strains harbored the slaA gene. Multilocus Sequence Typing (MLST) experiments demonstrated that the clone emm75/ST49/ smeZ, slaA was associated to OSAS cases. In conclusion, we found an association between slaA gene and the GAS OSAS strains, and we showed that the clone emm75/ST49 harboring genes smeZ and slaA was exclusively isolated from patients affected by OSAS, thus suggesting that this genotype might be associated to the pathogenesis of OSAS, although further studies are needed to elucidate the possible role of SlaA in tonsil hypertrophy development.

Paediatric obstructive sleep apnoea syndrome (OSAS) is associated with tonsil colonisation by Streptococcus pyogenes.

The involvement of pathogenic bacteria in obstructive sleep apnoea syndrome (OSAS) has yet to be elucidated. We investigated the possible role of group A streptococcus (GAS) in OSAS pathogenesis. In 40 tonsillectomized patients affected by OSAS and 80 healthy controls, significant (p < 0.0001) association of GAS with paediatric OSAS was found. Supernatant from streptolysin O (SLO)-producing GAS induced production of cysteinyl leukotrienes (CysLTs) in tonsil mononuclear cells (TMCs). CysLTs-treated TMCs showed significant (p < 0.05) proliferation of CD4+ T, CD19+ and CD19+CD27+CD38+ B lymphocytes. We discovered a SLO-dependent activation of CysLTs production through a pathway involving TOLL-like receptor 4 (TLR4), TIR-domain-containing adapter-inducing interferon-ß (TRIF), Myeloid differentiation primary response gene 88 (MyD88), and p38 MAP Kinase. In conclusion, we hypothesise that GAS may contribute to paediatric tonsillar hyperplasia through CysLTs production induced by SLO, and this might explain its association with OSAS.

Staphylococcus aureus-dependent septic arthritis in murine knee joints: local immune response and beneficial effects of vaccination.

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use.

Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF⁻ B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

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.

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.

Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria.

BACKGROUND: Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra), have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated. RESULTS: B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1beta upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1beta. CONCLUSIONS: A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.