Effectiveness of vaccination with recombinant HpaA from Helicobacter pylori is influenced by host genetic background

Several studies have explored the production and immunogenicity of HpaA as a potential protective antigen against Helicobacter pylori but little is known regarding its protective capabilities. We therefore evaluated the protective efficacy of recombinant HpaA (rHpaA) as a candidate vaccine antigen against H. pylori. To explore the impact of genetic diversity, inbred and outbred mice were prophylactically and therapeutically immunized with rHpaA adjuvanted with cholera toxin (CT). Prophylactic immunization induced a reduction in bacterial colonization in BALB/c and QS mice, but was ineffective in C57BL/6 mice, despite induction of antigen-specific antibodies. By contrast, therapeutic immunization was effective in all three strains of mice. Prophylactic immunization with CT-adjuvanted rHpaA was more effective when delivered via the nasal route than following intragastric delivery in BALB/c mice. However, HpaA-mediated protection was inferior to that induced by bacterial lysate. Hence, protective efficacy is inducible with vaccines containing HpaA, most relevantly shown in an outbred population of mice. The effectiveness of protection induced by HpaA antigen was influenced by host genetics and was less effective than lysate. HpaA therefore has potential for the development of effective immunization against H. pylori but this would probably entail the antigen to be one component of a multiantigenic vaccine.     

Unique mechanism of Helicobacter pylori for colonizing the gastric mucus

Helicobacter pylori is a human gastric pathogen causing chronic infection. Urease and motility using flagella are essential factors for its colonization. Urease of H. pylori exists both on the surface and in the cytoplasm, and is involved in neutralizing gastric acid and in chemotactic motility. H. pylori senses the concentration gradients of urea in the gastric mucus layer, then moves toward the epithelial surface by chemotactic movement. The energy source for the flagella movement is the proton motive force. The hydrolysis of urea by the cytoplasmic urease possibly generates additional energy for the flagellar rotation in the mucus gel layer.     

Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens

Mice and guinea pigs were intranasally immunized with either recombinant lipoprotein 20 or Helicobacter pylori outer membrane vesicles (OMV). Cholera toxin was used as mucosal adjuvant. In mice, both vaccines elicited systemic and local IgG responses, which correlated with significantly lower levels of H. pylori colonization. In contrast, only OMV proved immunogenic in guinea pigs, with the development of both systemic and local immune responses. These antibodies did not, however, correlate with protection in these animals, which suggests that vaccine formulation is as important as choice of antigen in the development of an H. pylori vaccine.     

Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility

Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a sigma(28)-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.     

Phasevarion mediated epigenetic gene regulation in Helicobacter pylori

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.     

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized. MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp. (H. pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp. (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp. flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods. RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e. lipid A, core and O-polysaccharide chains, were gained. All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H. mustelae and H. hepaticus produced truncated semirough lipopolysaccharides. However, in contrast to lipopolysaccharides of H. pylori and H. mustelae, no blood group mimicry was detected in the other Helicobacter spp. examined. Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus. Although shared lipopolysaccharide-core epitopes with H. pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp. The lipopolysaccharides of the gastric helicobacters, H. bizzozeronii and H. mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H. pylori lipopolysaccharide, whereas H. bilis, 'Helicobacter sp. flexispira' and H. hepaticus formed a cluster with approximately 1000-10,000-fold lower activities. H. pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H. pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp. were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation. CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches.     

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.     

Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy

In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.     

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.     

Inhibition of H. pylori colonization and prevention of gastritis in murine model

Helicobacter pylori is a Gram-negative spiral bacterium that colonizes human gastric mucosa causing infection. In this study aiming at inhibition of H. pylori infection we made an attempt to evaluate immunogenicity of the total (UreC) and C-terminal (UreCc) fragments of H. pylori urease. Total UreC and its C-terminal fragment were expressed in E. coli. Recombinant proteins were analyzed by SDS-PAGE and western blot and then purified by Ni-NTA affinity chromatography. Female C57BL6/j mice were immunized with the purified proteins (UreC and UreCc). Antibody titers from isolated sera were measured by ELISA. Immunized mice were then challenged by oral gavage with live H. pylori Sydney strain SS1. Total of 109 CFU were inoculated into stomach of immunized and unimmunized healthy mice three times each at one day interval. Eight weeks after the last inoculation, the blood sample was collected and the serum antibody titer was estimated by ELISA. Stomach tissues from control and experimental animal groups were studied histopathologically. UreC and UreCc yielded recombinant proteins of 61 and 31 kDa respectively. ELIZA confirmed establishment of immunity and the antibodies produced thereby efficiently recognized H. pylori and inhibited its colonization in vivo. Pathological analysis did not reveal established infection in immunized mice challenged with H. pylori. The results support the idea that UreC and UreCc specific antibodies contribute to protection against H. pylori infections.     

NikR-mediated regulation of Helicobacter pylori acid adaptation

Nickel is the cofactor of the Helicobacter pylori urease enzyme, a factor essential for the chronic colonization of the acidic hostile environment in the human stomach. The NikR regulatory protein directly controls urease expression and regulates the uptake of nickel, and is also able to regulate the expression of other regulatory proteins including the iron-responsive regulator Fur. Through regulatory crosstalk and overlapping regulons, the NikR protein controls the expression of many systems important for colonization and acid adaptation. Despite the paucity of regulatory proteins, this enables H. pylori to optimally adapt to conditions in the stomach, making it one of the most successful human pathogens.     

Helicobacter pylori motility

Motility is essential for Helicobacter pylori colonization. This review discusses the biochemistry, genetics and genomics of the H. pylori flagellum, and compares these features with well-characterized bacteria.     

Cell envelope characteristics of Helicobacter pylori: their role in adherence to mucosal surfaces and virulence

Abstract Helicobacter pylori colonises the gastric mucosa of humans and causes both antral gastritis and duodenal ulcer disease. Exactly how H. pylori causes disease is not known but several pathogenic determinants have been proposed for the organism. These include adhesins, cytotoxins and a range of different enzymes including urease, catalase and superoxide dismutase. Surface molecules of H. pylori such as flagella, lipopolysaccharide, the urease enzyme and outer membrane proteins are putative adhesin molecules. While phosphatidylethanolamine and the Lewis^b blood group antigen have been proposed as receptor molecules for the organism the exact mechanism by which H. pylori adheres to the gastric mucosa has still to be identified. Characterisation of the adhesins of H. pylori could lead to the development of adhesin analogues for use in the inhibition of colonisation and improved therapy for ulcer disease. In vivo studies with isogenic mutants which are incapable of adhering to the gastric mucosa would greatly clarify the significance of adherence. Such mutants could possibly be useful as a vaccine against infection with wild-type organisms.     

Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model

Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA(183-203)) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.