Helicobacter urease suppresses cytotoxic CD8+ T-cell responses through activating Myh9-dependent induction of PD-L1.

As a key virulence factor for persistent colonization, urease B subunit (UreB) is considered to be an ideal vaccine antigen against Helicobacter pylori infection. However, the role and molecular mechanisms of UreB involved in immune microenvironment dysregulation still remain largely unknown. In the present study, we evaluated the effects of UreB on macrophage activation and found that UreB induced PD-L1 accumulation on bone marrow-derived macrophages (BMDMs). Co-culture assays further revealed that UreB-induced PD-L1 expression on BMDMs significantly decreased the proliferation and secretion of cytolytic molecules (granzyme B and perforin) of splenic CD8+ T cells isolated from inactivated H. pylori-immunized mice. More importantly, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation techniques, it has been confirmed that myosin heavy chain 9 (Myh9) is a direct membrane receptor for UreB and is required for PD-L1 up-regulation on BMDMs. Molecular studies further demonstrated that the interaction between UreB and Myh9 decreased GCN2 autophosphorylation and enhanced the intracellular pool of amino acids, leading to the up-regulation of S6K phosphorylation, a commonly used marker for monitoring activation of mTORC1 signaling activity. Furthermore, blocking mTORC1 activation with its inhibitor Temsirolimus reversed the UreB-induced PD-L1 up-regulation and the subsequent inhibitory effects of BMDMs on activation of cytotoxic CD8+ T-cell responses. Overall, our data unveil a novel immunosuppressive mechanism of UreB during H. pylori infection, which may provide valuable clues for the optimization of H. pylori vaccine.

Helicobacter pylori adhesins: HpaA a potential antigen in experimental vaccines for H. pylori.

BACKGROUND: Helicobacter pylori is a gram-negative bacterium involved in many gastric pathologies such as ulcers and cancers. Although the treatment for this infection has existed for several years, the development of a vaccine is nevertheless necessary to reduce the severe forms of the disease. For more than three decades, many advances have been made particularly in the understanding of virulence factors as well as the pathogenesis of gastric diseases caused by H. pylori. Among these key virulence factors, specific antigens have been identified: Urease, Vacuolating cytotoxin A (VacA), Cytotoxin-associated gene A (CagA), Blood group antigen-binding adhesin (BabA), H. pylori adhesin A (HpaA), and others. OBJECTIVES: This review will focus on H. pylori adhesins, in particular, on HpaA and on the current knowledge of H. pylori vaccines. METHODS: All of the information included in this review was retrieved from published studies on H. pylori adhesins in H. pylori infections. RESULTS: These proteins, used in their native or recombinant forms, induce protection against H. pylori in experimental animal models. CONCLUSION: H. pylori adhesins are known to be promising candidate vaccines against H. pylori. Future research should be carried out on adhesins, in particular, on HpaA.

Oral immunization of mice with a multivalent therapeutic subunit vaccine protects against Helicobacter pylori infection.

Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.

Therapeutic Protection Against H. pylori Infection in Mongolian Gerbils by Oral Immunization With a Tetravalent Epitope-Based Vaccine With Polysaccharide Adjuvant.

Urease is an effective target for design of a therapeutic epitope vaccine against Helicobacter pylori (H. pylori). In our previous studies, an epitope vaccine CTB-UE containing Th and B epitopes from H. pylori urease was constructed, and the CTB-UE vaccine could provide therapeutic effect on H. pylori infection in mice. However, a multivalent vaccine, combining different antigens participating in different aspects of H. pylori colonization and pathogenesis, may be more effective as a therapeutic vaccine than a univalent vaccine targetting urease. Therefore, a multivalent epitope vaccine FVpE, containing Th1-type immune adjuvant NAP, three selected functional fragments from CagA and VacA, and an urease multi-epitope peptide (UE) from CTB-UE, was constructed in this study and expected to obtain better sterilizing immunity than the univalent epitope vaccine CTB-UE. The therapeutic effect of multivalent epitope vaccine FVpE with polysaccharide adjuvant (PA) was evaluated in H. pylori-infected Mongolian gerbil model. The results showed that both FvpE and CTB-UE vaccine could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect on H. pylori urease activity. However, only FVpE could induce high levels of specific antibodies to CagA, VacA, and NAP. In addition, oral therapeutic immunization with FVpE plus PA significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils compared with oral immunization with CTB-UE plus PA, or FVpE only, and the FVpE vaccine with PA even exhibited sterilizing immunity. The protection of FVpE was related to the mixed CD4+ T cell responses and epitope-specific antibodies against various H. pylori antigens. These results indicate that a multivalent epitope vaccine targetting various H. pylori antigens could be a promising candidate against H. pylori infection.

Neutralizing antibodies for Helicobacter pylori urease inhibit bacterial colonization in the murine stomach in vivo.

Helicobacter pylori (H. pylori) urease is a key protein for persistent infection of the bacteria in the stomach. Although H. pylori generally induce anti-H. pylori-specific antibodies (Abs), these Abs do not usually work for eradication or prevention of the H. pylori infection. In our previous study, we identified a linear epitope composed of 19-mer peptides termed UB-33, CHHLDKSIKEDVQFADSRI, within the large subunit of H. pylori urease. Anti-UB-33-specific Abs neutralized the enzymatic activity of H. pylori urease in vitro. In the present study, we evaluated the effect of immunization of BALB/c mice with H. pylori UB-33 peptide. After confirming the production of anti-UB-33-specific Abs, mice were challenged orally with H. pylori Sydney Strain-1 (SS-1). Mice producing anti-UB-33-specific Abs were not infected with SS-1, and the amount of SS-1 isolate in their stomach was significantly reduced. Also, the urease-negative mutant of H. pylori, HPP1801, did not colonize in the stomach, indicating that H. pylori urease was a critical element for infection of H. pylori in the gastric mucosa. Moreover, mice producing UB-33-specific Abs apparently suppressed H. pylori infection in the stomach where anti-UB-33 Abs were secreted in the gastric juice, indicating that H. pylori colonization was inhibited in the presence of anti-UB-33 Abs. In addition, the neutralization activity of sera from mice immunized with purified urease was less potent than that in the sera from mice immunized with UB-33. Furthermore, the recognition of epitope UB-33 was mediated through Toll-like receptor 2 (TLR2) on the B-1 cells using TLR2-knockout BALB/c mice in vivo. These results indicate that liner peptide UB-33 should be used for immunization to induce neutralizing Abs instead of purified H. pylori urease to prevent H. pylori infection and their colonization in the stomach.

Synergistic effect of anti-Helicobacter pylori urease immunoglobulin Y from egg yolk of immunized hens and Lactobacillus johnsonii No.1088 to inhibit the growth of Helicobacter pylori in vitro and in vivo.

Helicobacter pylori is a pathogenic bacterium that infects the stomach, causing chronic gastritis; and it is also considered to be related to the occurrence of gastric cancers. Although some eradication regimens including multiple antibiotics have been developed, the emergence of resistance to antibiotics becomes problematic. Therefore, other approaches to compensate or augment the effects of standard regimens are needed. In this study, we examined the possible synergistic effects of anti-H. pylori urease IgY and Lactobacillus johnsonii No.1088 (LJ88) both in vitro and in vivo. Anti-H. pylori urease IgY was purified from egg yolks laid by the hens immunized with urease purified from H. pylori. LJ88 is a unique strain of lactic acid bacterium isolated from human gastric juice, and it has been reported to inhibit H. pylori both in vitro and in vivo. The in vitro mixed culture study showed that anti-H. pylori urease IgY augmented the anti-H. pylori activity of LJ88 against both clarithromycin-sensitive and -resistant H. pylori strains. In a germ-free mice infection model, combined administration of daily anti-H. pylori urease IgY and weekly living LJ88 significantly reduced H. pylori infections, whereas either monotherapy did not. In an in vivo human gut microbiota-associated mice model, not only daily administration of living LJ88 but also heat-killed one significantly reduced an H. pylori infection in the stomach when combined with anti-H. pylori urease IgY. The extent of reduction of the stomach H. pylori by such a combination therapy was larger than that reported for LJ88 monotherapy. These results taken together revealed a synergistic effect of anti-H. pylori urease IgY and living or heat-killed LJ88, thus suggesting that such a combination might be a promising therapy to possibly compensate and/or augment standard anti-H. pylori regimens.

Selection of a fully human single domain antibody specific to Helicobacter pylori urease.

Helicobacter pylori bacteria are involved in gastroduodenal disorders, including gastric adenocarcinoma. Since the current therapies encounter with some significant shortcomings, much attention has been paid to the development of new alternative diagnostic and treatment modalities such as immunomedicines to target H. pylori. Having used phage display technology, we isolated fully humane small antibody (Ab) fragment (VL) against the Flap region of urease enzyme of H. pylori to suppress its enzymatic activity. Solution biopanning (SPB) and screening process against a customized biotinylated peptide corresponding to the enzyme Flap region resulted in the selection of VL single domain Abs confirmed by the enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. The selected Ab fragments showed a high affinity with a KD value of 97.8 × 10-9 and specificity to the enzyme with high inhibitory impact. For the first time, a VL single domain Ab was isolated by SPB process against a critical segment of H. pylori urease using a diverse semi-synthetic library. Based on our findings, the selected VL Ab fragments can be used for the diagnosis, imaging, targeting, and/or immunotherapy of H. pylori. Further, Flap region shows great potential for vaccine therapy.

The mucosal adjuvant effect of plant polysaccharides for induction of protective immunity against Helicobacter pylori infection.

Some plant polysaccharides (PPSs) had been used as the adjuvants for systemic vaccination. In this study, we investigated whether PPSs could exhibit adjuvant effect at the mucosa. Groups of mice were intranasally immunized with Epimedium Polysaccharide (EPS), Trollius chinensis polysaccharide (TCPS), Siberian solomonseal rhizome polysaccharide (SSRPS) and Astragalus polysaccharides (APS) together with ovalbumin (OVA). Significantly higher levels of OVA-specific IgG in serum and secretory IgA in saliva, vaginal wash and intestinal lavage fluid were induced after immunization with OVA plus one of the four PPSs compared to OVA alone. Antigen absorption and TLR2 (Toll-like receptor 2) activation may be related to their mucosal adjuvant effect. Of note, when APS used as an adjuvant, intranasally vaccination with recombination UreB (rUreB, Urease subunit B) conferred more robust protection against Helicobacter pylori (H. pylori). Immunized with rUreB in combination APS resulted in mixed specific Th1 and Th17 immune response, which may contribute to the inhibition of H. pylori colonization. Though specific Th2-dominant responses were elicited when the other three PPS intranasally immunized with rUreB, no significant difference in the protective effect were found between those groups and rUreb alone group. Taken together, the four PPSs may be promising candidates for mucosal adjuvant, and APS could enhance rUreB-specific protective immunity against H. pylori infection.

Protection Against Helicobacter pylori Infection in BALB/c Mouse Model by Oral Administration of Multivalent Epitope-Based Vaccine of Cholera Toxin B Subunit-HUUC.

Vaccination is an increasingly important alternative approach to control Helicobacter pylori infection, since H. pylori resistance to previously efficacious antibiotic regimens is increased, and H. pylori eradication treatment for upper gastrointestinal diseases is becoming less successful. Fortunately, an efficient oral monovalent H. pylori vaccine has been developed. However, compared with monovalent vaccines, multivalent vaccines have the potential to induce more effective and comprehensive protection against H. pylori infection. In this study, we designed and produced a multivalent epitope-based vaccine cholera toxin B subunit (CTB)-HUUC with the intramucosal adjuvant CTB and tandem copies of B-cell epitopes (HpaA132-141, UreA183-203, and UreB321-339) and T-cell epitopes (HpaA88-100, UreA27-53, UreB229-251, UreB317-329, UreB373-385, UreB438-452, UreB546-561, CagA149-164, and CagA196-217) from H. pylori adhesion A subunit (HpaA), urease A subunit (UreA), urease B subunit (UreB), and cytotoxin-associated antigen (CagA). Serum IgG, stomach, and intestine mucosal sIgA from mice after CTB-HUUC vaccination neutralized H. pylori urease activity in vitro. CTB-HUUC vaccination promoted H. pylori-specific lymphocyte responses and a mixed CD4+ T cell immune response as indicated by IFN-γ, interleukin-4, and interleukin-17 production in mice. Both oral prophylactic and therapeutic CTB-HUUC vaccinations reduced gastric urease activity and H. pylori infection and protected stomachs in mice. Taken together, CTB-HUUC is a promising potent and safe multivalent vaccine in controlling H. pylori infection in BALB/c mouse model.

A survey on chimeric UreB229-561-HpaA protein targeting Helicobacter pylori: Computational and in vitro urease activity valuation.

Helicobacter pylori (H. pylori) as microaerophilic, Gram-negative bacterium colonize the human gastric milieu, where it impetuses chronic disorders. Vaccination is a complementary plan, along with antibiotic therapy, for clearance of H. pylori. Today, Computer based tools are essential for the evaluation, design, and experiment for novel chimeric targets for immunological administration. The purpose of this experiment was immunoinformatic analysis of UreB and HpaA molecules in a fusion arrangement and also, construction and expression of recombinant protein containing chimeric sequences. The targets sequences were screened by using of standard in silico tools and immunoinformatic web servers. The high-resolution 3D models of the protein were created and were validated; indeed, the B-and T-cell restricted epitopes were mapped on the chimeric protein. The recombinant protein in frame of the expression vector pET28a were expressed and purified successfully. The urease activity and immunoblotting were performed in vitro condition. This study confirmed that the engineered protein as a highly conserved, hydrophilic, non-allergenic contained remarkable B-cell and T-cell epitopes. It was magnificently attained; chimeric UreB229-561-HpaA could provoke both humoral and cellular immunity. The immunoblotting was shown that the chimeric protein could be detected by serum of immunized animal and H.pylori positive patients. In this study, several antigenic patches from UreB and HpaA were identified that could be an efficient immune system activator. The in vitro analysis of our chimeric molecule confirmed its urease activity. It also confirmed that the chimeric protein could be detected by serum of immunized animal and H.pylori positive patients.

Intraperitoneal immunization with Urease loaded N-trimethyl Chitosan nanoparticles elicits high protection against Brucella melitensis and Brucella abortus infections.

Brucella (B) species are brucellosis causative agents, a worldwide zoonotic illness causing Malta fever in humans and abortion in domestic animals. In this work, we evaluated the vaccine potential of Trimethyl chitosan (TMC) nanoparticles formulation of Urease (TMC/Urease) against brucellosis. TMC/Urease nanoparticles and urease without any adjuvant were separately administered both orally and intraperitoneally. Intraperitoneal (i.p.) administration of urease alone as well as oral administration of both TMC/Urease nanoparticles and urease alone, elicited low titers of specific immunoglobulin G (IgG), while i.p. immunization with TMC/Urease nanoparticles induced high specific IgG production levels. As it was indicated by the cytokine assay and the antibody isotypes, i.p. immunization by urease alone, and TMC/Urease nanoparticles induced a mixed Th1-Th2 immune response, whereas oral administration of both urease alone and TMC/Urease nanoparticles induced a mixed Th1-Th17 immune response. In lymphocyte proliferation assay, spleen cells from i.p.-vaccinated mice with TMC/Urease nanoparticles showed a strong recall proliferative response. Vaccinated animals were challenged with virulent strains of B. melitensis and B. abortus. I.p. vaccination with TMC/Urease nanoparticles resulted in a high degree of protection. Altogether, our results indicated that TMC nanoparticles are a potent delivery system for i.p.-administered Brucella antigens.

Nongenetically modified Lactococcus lactis-adjuvanted vaccination enhanced innate immunity against Helicobacter pylori.

BACKGROUND: Gram-positive enhancer matrix particles (GEM) produced by Lactococcus lactis can enhance vaccine-induced immune response. However, the mechanism under which this adjuvant mounts the efficacy of orally administered vaccines remains unexplored. MATERIALS AND METHODS: We used a prophylactic mice model to investigate the mechanism of GEM-adjuvanted vaccination. Helicobacter pylori urease-specific antibody response was monitored and detected in murine serum by ELISA. Urease-specific splenic cytokine profile was examined. Gastric inflammatory responses were measured on day 43 or 71 by quantitative real-time PCR, flow cytometry and histology. RESULTS: We found that GEM enhanced the efficiency of oral H. pylori vaccine by promoting innate immunity. The vaccine CUE-GEM composed of GEM particles and recombinant antigen CTB-UE provided protection of immunized mice against H. pylori insult. The protective response was associated with induction of postimmunization gastritis and local Th1/Th17 cell-medicated immune response. We showed that innate inflammatory responses including neutrophil chemokines CXCL1-2, neutrophils, and antimicrobial proteins S100A8 and MUC1 were significantly elevated. Within all infected mice, S100A8 and MUC1 levels were negatively correlated with H. pylori burden. Strikingly, mice receiving GEM also show reduction of colonization, possibly through natural host response pathways to recruit CD4+ T cells and promote S100A8 expression. CONCLUSIONS: These findings suggest that GEM-based vaccine may impact Th1/Th17 immunity to orchestrate innate immune response against H. pylori infection.

Oral Immunization with a Multivalent Epitope-Based Vaccine, Based on NAP, Urease, HSP60, and HpaA, Provides Therapeutic Effect on H. pylori Infection in Mongolian gerbils.

Epitope-based vaccine is a promising strategy for therapeutic vaccination against Helicobacter pylori (H. pylori) infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori, remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP), heat shock protein 60 (HSP60) and H. pylori adhesin A (HpaA) was constructed based on mucosal adjuvant cholera toxin B subunit (CTB), Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes (UreA27-53, UreA183-203, HpaA132-141, and HSP60189-203), and also the epitope-rich regions of urease B subunit (UreB158-251 and UreB321-385) predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori-infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes (UreB158-172, UreB181-195, UreB211-225, UreB349-363, HpaA132-141, and HSP60189-203). In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4+ T cell (Th cell) response, IgG, and secretory IgA (sIgA) antibodies to H. pylori. These results indic ate that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.

Immunologic properties and therapeutic efficacy of a multivalent epitope-based vaccine against four Helicobacter pylori adhesins (urease, Lpp20, HpaA, and CagL) in Mongolian gerbils.

BACKGROUND: Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and adhesins, which are on the surface of H. pylori, play a pivotal role in binding to human gastric mucosa. MATERIALS AND METHODS: In the present study, we constructed a multivalent epitope-based vaccine named CFAdE with seven carefully selected antigenic fragments from four H. pylori adhesins (urease, Lpp20, HpaA and CagL). The specificity, immunogenicity and ability to produce neutralizing antibodies of CFAdE were evaluated in BALB/c mice. After that, its therapeutic efficacy and protective immune mechanisms were explored in H. pylori-infected Mongolian gerbils. RESULTS: The results indicated that CFAdE could induce comparatively high levels of specific antibodies against urease, Lpp20, HpaA and CagL. Additionally, oral therapeutic immunization with CFAdE plus polysaccharide adjuvant (PA) significantly decreased H. pylori colonization compared with oral immunization with urease plus PA, and the protection was correlated with IgG and sIgA antibody and antigen-specific CD4+ T cells. CONCLUSIONS: This study indicated that the multivalent epitope-based vaccine, which targeted multiple adhesins in adherence of H. pylori to the gastric mucosa, is more effective than the univalent vaccine targeting urease only. This multivalent epitope-based vaccine may be a promising therapeutic candidate vaccine against H. pylori infection.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants.

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease ß subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

A novel design of a multi-antigenic, multistage and multi-epitope vaccine against Helicobacter pylori: An in silico approach.

Helicobacter pylori have colonized the gastric mucosa of half of the population worldwide. This bacterium is classified as a definitive type I carcinogen by the World Health Organization and no effective vaccine has been found against it yet. Thus, a logical and rational vaccine design against H. pylori is necessary. Because of its tremendous complexity and elicited immune responses, the vaccine design should considered multiple antigens to enhance immune-protection, involved in the different stages of pathogenesis besides inducing a specific immune response by B- and T-cell multi-epitopes. In this study, emphasis was placed on the design of a new unique vaccine named CTB-multiHp. In silico techniques were used to design a chimeric construct consisting of cholera toxin B subunit fused to multi-epitope of urease B (residue 148-158, 188-198), cytotoxin-associated gene A (residue 584-602), neutrophil activating protein (residue 4-28), vacuolating cytotoxin gene A (residue 63-81), H. pylori adhesine A (residue77-99), heat shock protein A (residue 32-54) and gamma glutamyl transpeptidase (residue 271-293). The tertiary structure and features of the vaccine were analyzed. The chimeric protein was expressed in Escherichia coli BL21 and the serology analyses indicated that the CTB-multiHp protein produced exhibit immune-reactivity. The results showed that CTB-multiHp could be a good vaccine candidate against H. pylori. Ongoing studies will evaluate the effects of CTB-multiHp against H. pylori infection.

Decreasing prevalence of Helicobacter pylori according to birth cohorts in urban China.

BACKGROUND/AIMS: The Helicobacter pylori prevalence has been decreasing in many parts of the world. This study aimed to investigate the current epidemiological status of H. pylori infection in urban China. MATERIALS AND METHODS: The study included 51,299 subjects aged ≥18 years who underwent health checkups between April 2013 and June 2016 in a city of China. H. pylori infection was determined by detecting H. pylori urease-IgG antibodies. Statistical analyses included chi-square tests for trends and curve fitting. RESULTS: The overall H. pylori prevalence was found to be 31.9%, with the highest prevalence in the 1950-1959 birth cohort. It was lower in the subsequent birth cohorts (trends, p<0.001). The decrease in H. pylori prevalence was correlated with the increase in per capita gross domestic product (GDP) and real per capita GDP; the power model was best fitted (R2=0.914 and 0.997 and p=0.011 and 0.000, respectively). CONCLUSION: There has been a striking decrease in the H. pylori prevalence in urban China. The birth cohort effect and economic growth are the most likely causes of this phenomenon.

IL-22-induced antimicrobial peptides are key determinants of mucosal vaccine-induced protection against H. pylori in mice.

Despite the recent description of the mucosal vaccine-induced reduction of Helicobacter pylori natural infection in a phase 3 clinical trial, the absence of immune correlates of protection slows the final development of the vaccine. In this study, we evaluated the role of interleukin (IL)-22 in mucosal vaccine-induced protection. Gastric IL-22 levels were increased in mice intranasally immunized with urease+cholera toxin and challenged with H. felis, as compared with controls. Flow cytometry analysis showed that a peak of CD4+IL-22+IL-17+ T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice. The inhibition of the IL-22 biological activity prevented the vaccine-induced reduction of H. pylori infection. Remarkably, anti-microbial peptides (AMPs) extracted from the stomachs of vaccinated mice, but not from the stomachs of non-immunized or immunized mice, injected with anti-IL-22 antibodies efficiently killed H. pylori in vitro. Finally, H. pylori infection in vaccinated RegIIIß-deficient mice was not reduced as efficiently as in wild-type mice. These results demonstrate that IL-22 has a critical role in vaccine-induced protection, by promoting the expression of AMPs, such as RegIIIß, capable of killing Helicobacter. Therefore, it can be concluded that urease-specific memory Th17/Th22 cells could constitute immune correlates of vaccine protection in humans.

Recombinant Bacillus subtilis spores expressing cholera toxin B subunit and Helicobacter pylori urease B confer protection against H. pylori in mice.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The limitations of current therapies for H. pylori infection include poor compliance and antibiotic resistance. Therefore, an effective anti-H. pylori vaccine would be an alternative or complement to antibiotic treatment. Urease B (UreB) is considered an ideal vaccine antigen against H. pylori infection. In this study, cholera toxin B subunit (CTB), a mucosal adjuvant, was used to enhance the immunogenicity of a novel Bacillus subtilis spore vaccine expressing CTB-UreB, along with the B. subtilis spore coat protein CotC as a fusion protein. Oral administration of B. subtilis spores expressing CotC-UreB or CotC-CTB-UreB led to increased levels of UreB-specific IgG in serum and UreB-specific IgA in faeces, as well as elevated levels of IL-10 and IFN-γ in splenocytes. In addition, oral administration of CotC-UreB or CotC-CTB-UreB spores induced significant reductions (80.0 and 90.5 %, respectively) in gastric H. pylori bacterial load (1.11±0.36×105 and 0.53±0.21×105 c.f.u., respectively) compared to that of the CotC control group (5.56±1.64×105 c.f.u., P<0.01). Moreover, CotC-CTB-UreB spores were significantly more effective at reducing the bacterial load than CotC-UreB spores (P<0.05). These results indicate that CotC-CTB-UreB-expressing B. subtilis spores are a potential vaccine candidate for the control of H. pylori infection.

Fast and sensitive detection of foodborne pathogen using electrochemical impedance analysis, urease catalysis and microfluidics.

Early screening of pathogenic bacteria is a key to prevent and control of foodborne diseases. In this study, we developed a fast and sensitive bacteria detection method integrating electrochemical impedance analysis, urease catalysis with microfluidics and using Listeria as model. The Listeria cells, the anti-Listeria monoclonal antibodies modified magnetic nanoparticles (MNPs), and the anti-Listeria polyclonal antibodies and urease modified gold nanoparticles (AuNPs) were incubated in a fluidic separation chip with active mixing to form the MNP-Listeria-AuNP-urease sandwich complexes. The complexes were captured in the separation chip by applying a high gradient magnetic field, and the urea was injected to resuspend the complexes and hydrolyzed under the catalysis of the urease on the complexes into ammonium ions and carbonate ions, which were transported into a microfluidic detection chip with an interdigitated microelectrode for impedance measurement to determine the amount of the Listeria cells. The capture efficiency of the Listeria cells in the separation chip was ∼93% with a shorter time of 30min due to the faster immuno-reaction using the active magnetic mixing. The changes on both impedance magnitude and phase angle were demonstrated to be able to detect the Listeria cells as low as 1.6×10(2)CFU/mL. The detection time was reduced from original ∼2h to current ∼1h. The recoveries of the spiked lettuce samples ranged from 82.1% to 89.6%, indicating the applicability of this proposed biosensor. This microfluidic impedance biosensor has shown the potential for online, automatic and sensitive bacteria separation and detection.