Laura Pancotto

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.

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.

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

ABSTRACT 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.

Immunohistochemical Study of Lymphocyte Populations Infiltrating the Gastric Mucosa of Beagle Dogs Experimentally Infected with Helicobacter pylori

ABSTRACT Experimental infection of beagle dogs with Helicobacter pylori induces recruitment to the gastric mucosae of neutrophils at early stages and later of mononuclear cells that organize into lymphoid follicles. These structures become macroscopically evident and consist of peripheral CD4+ T lymphocytes and central CD21+ B lymphocytes. Furthermore, transient expression of interleukin-8 (IL-8) parallels the presence of neutrophils in the gastric mucosae, whereas expression of IL-6 tends to persist chronically.

Polyphenols Reduce Gastritis Induced by Helicobacter pylori Infection or VacA Toxin Administration in Mice

ABSTRACT Helicobacter pylori colonizes the human gastric mucosa, causing inflammation that leads to atrophic gastritis, and it can cause peptic ulcer and gastric cancer. We show that polyphenol administration to mice experimentally infected by H. pylori or treated with VacA toxin can limit gastric epithelium damage, an effect that may be linked to VacA inhibition.

Sortase A confers protection against Streptococcus pneumoniae in mice.

ABSTRACT 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.

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.

Helicobacter pylori Infection Negatively Influences Pregnancy Outcome in a Mouse Model

Background.  Helicobacter pylori infects the human stomach, causing gastritis, peptic ulcer, and gastric cancer. H. pylori infection has also been related to extra‐gastric disorders. We investigated whether H. pylori infection can influence pregnancy in a murine model.

Expression of the Streptococcus pneumoniae pilus-1 undergoes on and off switching during colonization in mice.

Streptococcus pneumoniae pili contribute to adherence and virulence. The regulation of pilus-1 expression is bistable, thus piliated strains contain a variable proportion of pilus-1-non-expressing bacteria. We investigated whether such proportion changes during colonization. Pilus-1-expressing bacteria were quantified in nasopharyngeal washes and pharyngeal tissues from mice that received intranasally bacterial populations with high (H), medium (M) or low (L) pilus-1 expression rates. In nasopharyngeal washes, at early colonization stages, pilus-1 expression rates decreased in H population, while increased in L and M; at later stages, expression rates decreased or remained low. Similar trends were observed in pharyngeal tissues, where, however, at late stages the expression rates were medium-high. In conclusion, pilus-1 is preferentially expressed at early colonization stages, consistently with its role in adhesion, while at later stages the expression is partially switched off. Pilus-1 expression rates observed in clinical isolates in vitro may not reflect the actual rates during colonization/infection.

Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes.

Helicobacter pylori is a Gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1β, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other Gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.