Safety and efficacy of E coli enterotoxin adjuvant for urease-based rectal immunization against Helicobacter pylori

Low dose E. coli heat-labile enterotoxin (LT), delivered orally or enterically, has been used as an adjuvant for Helicobacter pylori (H. pylori) urease in healthy adults. In this study we aim to test the safety and adjuvant efficacy of LT delivered rectally together with recombinant H. pylori urease. Eighteen healthy adults without present or past H. pylori infection were enrolled in a double blind, randomized, ascending dose study to receive either urease (60 mg), or urease (60 mg) + LT (5 or 25 microg). The immunization preparation was administered per rectum on days 0, 14 and 28. Serum, stool and saliva anti-urease and anti-LT IgG and IgA antibodies (Abs) were measured and urease-specific and LT-specific antigen secreting cells (ASCs) were counted in peripheral blood at baseline and 7 (ASC counts) or 14 days (antibody levels) after each dosing. Peripheral blood lymphoproliferation assays were also performed at baseline and at the end of the study.Rectally delivered urease and LT were well tolerated. Among the 12 subjects assigned to urease+LT, 2 (16.7%) developed anti-urease IgG Abs, 1 (8.3%) developed anti-urease IgA Abs, and 3 (25%) showed urease-specific IgA(+) ASCs. Immune responses to LT were more vigorous, especially in subjects exposed to 5 microg LT. In the urease+ 5 microg LT group, anti-LT IgG and IgA Abs developed in 60 and 80% of the subjects, respectively, while LT-specific IgG(+) and IgA(+) ASCs were detected in all subjects. The magnitude of the anti-LT response was much higher than the response to urease. No IgA anti-urease or anti-LT Abs were detected in stool or saliva and lymphocyte proliferative responses to urease were unsatisfactory. In conclusion, rectal delivery of 5 microg LT is safe and induces vigorous systemic anti-LT immune responses. Further studies are needed to determine if LT can be an effective adjuvant for rectally delivered antigens.     

Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori.

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively). The mucosal prime-parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.     

Immunization with recombinant Helicobacter pylori urease decreases colonization levels following experimental infection of rhesus monkeys

Rhesus monkeys, naturally colonized with H. pylori as indicated by culture and histology were immunized with either 40 mg recombinant H. pylori urease administered orally together with 25 microg Escherichia coli heat-labile enterotoxin (LT) or immunized with LT alone. An initial 6 doses were administered over an 8 week period. All five vaccinated monkeys had a greater than two-fold rise in urease-specific serum IgG and IgA level and urease-specific salivary IgA was induced in 3 of 5 vaccinated animals after 6 or 7 doses of vaccine. Vaccination had no measurable therapeutic effect on H. pylori colonization. H. pylori was eradicated from these monkeys with a course of antimicrobials plus omeprazole, a 7th vaccine dose was given (10 months after the 6th dose) and they were rechallenged with H. pylori. Necropsy was performed 23 weeks after rechallenge and H. pylori colonization was determined by histological examination of 12 individual gastric sites. A significant reduction in colonization (p < or = 0.0001; Friedman's analysis of variance) was found in the vaccinated animals. Histopathologic examination of necropsy tissues also revealed a trend towards reduced gastritis and epithelial alterations in the vaccinated group compared to animals receiving LT alone. This study provides the first evidence for effective vaccination of nonhuman primates against H. pylori, and preliminary evidence that a reduction in bacterial density attributable to immunization may lessen gastric inflammation.     

Further development of the Helicobacter pylori mouse vaccination model

Immunisation against Helicobacter infection in mouse models has thus far produced neither complete protection against the bacteria, nor a complete prevention of the associated gastritis. This study aimed firstly to compare the sensitivities of the various methods used to assess H. pylori infection in the mouse model, and secondly to develop the experimental design to induce a more effective immunity, aimed at further reducing bacterial burden in the gastric tissue. Various mouse strains were prophylactically immunised with whole bacterial sonicate and cholera toxin before challenge with H. pylori-SS1. The relative sensitivities of the urease assay, histological assessment and the colony forming assay to detect levels of H. pylori colonisation were compared. Comparisons of different antigen doses and different timecourses of immunisation were performed. The colony forming assay was found to be far more sensitive than either the urease assay or histological assessment for determining the protective efficacies of immunisation. Mice which had 10(5) H. pylori per gram of stomach by colony assay were negative by histology and urease. Lower doses of whole cell sonicate were more protective than high doses and more effective immunisation was achieved by leaving at least 3 weeks between immunisation instead of weekly immunisations. In conclusion, for assessment of H. pylori colonisation in the mouse model, the colony forming assay should be used. The experimental protocol for immunisation has been altered to produce a significant improvement in protection. However, full protection has still not yet been achieved and more work is still required.     

Immune responses in mice to intranasal and intracutaneous administration of a DNA vaccine encoding Helicobacter pylori-catalase

We previously reported that the intracutaneous injection of DNA vaccines encoding Helicobacter pylori heat shock proteins elicited specific immune responses, and led to reduced infection in mice. In this study, we constructed DNA vaccine encoding H. pylori-catalase (pcDNA3.1-kat) and investigated the immune responses to intranasal and intracutaneous administration of pcDNA3.1-kat. C57/BL6 mice were immunized intracutaneously with 10 microg of pcDNA3.1-kat or intranasally with 50 microg of pcDNA3.1-kat. Catalase-specific IgG antibody was detected in the sera of intranasal and intracutaneous immunized mice. Both intranasal and intracutaneous immunized mice were significantly protected from colonization by H. pylori and had significantly reduced degrees of gastritis. These results demonstrate that DNA vaccine encoding H. pylori-catalase can induce an immune response against H. pylori, and that intranasal immunization works as well as intracutaneous immunization.     

Protective immunization against "Candidatus Helicobacter suis" with heterologous antigens of H. pylori and H. felis

"Helicobacter (H.) heilmannii" type 1 colonizes the human stomach. It has been shown to be identical to "Candidatus H. suis", a Helicobacter species colonizing the stomach of more than 60% of slaughter pigs. This bacterium is, until now, not isolated in vitro. The effect of vaccination on "Candidatus H. suis" infection was studied in a mouse model. Mice were vaccinated intranasally or subcutaneously with whole bacterial cell lysate of Helicobacter pylori or Helicobacter felis and subsequently challenge infected with "Candidatus H. suis". Intranasal and subcutaneous immunisation caused a decrease in faecal excretion of "Candidatus H. suis" DNA. Urease tests on stomach tissue samples at 16 weeks after challenge infection were negative in all H. felis intranasally immunized animals and in the majority of the animals of the other immunisation groups. Since PCR on stomach tissue samples at 16 weeks after challenge infection could still detect "Candidatus H. suis DNA" in all immunisation-challenge groups, complete clearance of challenge bacteria was not achieved.     

Transmission of Helicobacter spp. A challenge to the dogma of faecal-oral spread

Faecal oral spread is claimed by many to be the mode of transmission of the gastric pathogen Helicobacter pylori. This idea is based not on experimental data but because the epidemiology of H. pylori infection resembles that of other pathogens known to be spread by the faecal-oral route. This is in spite of the observation that no-one has been successful in culturing H. pylori from human stool. In this study, a series of transmission experiments are reported on animals infected with the gastric spirilla, Helicobacter felis and 'Gastrospirillum hominis'. Germfree mice and rats infected with H. felis did not transmit their infection to uninoculated mice despite prolonged contact in the same cage nor could the bacterium be isolated from their intestinal contents. This was confirmed in specific pathogen free mice where infected dams did not pass the helicobacter to their progeny. Similarly, mice infected with a human isolate of 'Gastrospirillum hominis' did not transmit the infection while in close contact with uninoculated mice. In contrast, in a limited series of experiments, both H. pylori and H. felis were transmitted from infected gnotobiotic Beagle puppies to uninfected animals in the same enclosure. In addition, the gastric mucus from a cat with indigenous 'Gastrospirillum'-like organisms was infectious for mice, whereas faecal content from the same animal was not. It is suggested that the difference between the murine and canine experiments is that the dogs are more likely to have oral-oral contact than rodents. Unlike dogs, mice and rats do not vomit and are coprophagous. It is concluded that the case for faecal-oral spread of Helicobacter species is 'not proven' and that the inter-oral route is more likely.     

A plasmid immunization construct encoding urease B of Helicobacter pylori induces an antigen-specific antibody response and upregulates the expression of beta-defensins and IL-10 in the stomachs of immunized mice

The objectives of this study were to investigate the efficacy of a prototype DNA immunization construct encoding the urease B subunit enzyme of Helicobacter pylori (H. pylori) for inducing adaptive and innate immune responses in mice immunized via intramuscular or subcutaneous routes and to further explore the adjuvant effects of the CpG motifs in the vector. Antibody, cytokine, and beta-defensin profiles were assessed in the stomachs of immunized animals: experiments were terminated 3 months after immunization because there was a significant increase in the anti-H. pylori urease B antibody response at Week 6 in mice immunized with the urease B construct. A long lasting expression of IL-10 mRNA was noted. Furthermore, a marked and sustained increase in the mRNA expression of beta-defensins was also observed, particularly beta1. This study demonstrates that an H. pylori urease B DNA construct can induce innate as well as adaptive immune responses in the stomachs of immunized mice. Upregulation of beta-defensin gene expression followed immunization and we believe that this is the first report of a DNA vaccine inducing innate anti-microbial responses. Such complex molecular interactions that modulate both innate and adaptive immune responses may be of critical importance in the control of mucosal pathogens, such as H. pylori.     

Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine

Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection.     

The attenuated Salmonella vaccine approach for the control of Helicobacter pylori-related diseases

The Gram-negative bacterium Helicobacter pylori is a widespread human pathogen that colonizes the gastric mucosa and is associated with gastro-intestinal illnesses such as gastritis, peptic ulcer, gastric lymphoma and gastric cancer. Current pharmacological therapies are becoming less reliable for the control of H. pylori due to the elevated costs and to the increasing number of antibiotic resistant strains. New vaccination strategies utilizing H. pylori antigens combined with adjuvants or delivery of antigens by attenuated Salmonella strains have been successful in protecting mice against H. pylori infections. Oral immunization with single doses of urease-expressing Salmonella vaccine strains elicits mucosal and systemic antibody responses and fully protects different mouse strains against challenge infections with H. pylori. The high efficacy in the mouse model, combined with remarkable immunogenicity, safety and low-cost production, makes attenuated live recombinant Salmonella promising vaccine candidates for the control of H. pylori-related diseases in humans.     

Immunisation against Helicobacter felis infection protects against the development of gastric MALT Lymphoma

The formation of mucosa-associated lymphoid tissue (MALT) in response to Helicobacter pylori infection is closely associated with the development of primary gastric MALT lymphoma. AIM: To examine whether immunisation against Helicobacter felis can protect against development of MALT lymphoma. RESULTS: The majority of control infected mice demonstrated MALT formation (13/15) and five developed lymphoma. Fifteen immunised mice were protected against bacterial challenge, of which only five had evidence of MALT formation and none developed lymphoma. Interestingly, of the four mice in which immunisation failed, all developed MALT and two of these had lymphoma. CONCLUSION: Effective immunisation against Helicobacter infection can protect against gastric MALT lymphoma. To our knowledge this is the first demonstration of vaccination protecting against a bacteria-induced malignancy.     

B cell responses in gastric antrum and duodenum following oral inactivated Helicobacter pylori whole cell (HWC) vaccine and LT(R192G) in H pylori seronegative individuals

To investigate whether B cell-specific responses could be elicited in the gastric mucosa of Helicobacter pylori (HP) naive subjects, five volunteers ingested three doses of a HP killed whole cell (HWC) vaccine with 25 microg of recombinant heat-labile toxin (LT(R192G)). Two of three subjects had detectable LT(R192G) and HWC IgA antibody secreting cell (ASC) gastric responses. LT(R192G) and HWC responses in duodenal were 5-14-fold higher than those detected in antral biopsies (P<0.01 and P=0.05, respectively). These results provide the first evidence that specific gastric B cell responses can be induced in HP-non-infected individuals following oral immunization.     

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease protect mice against H. pylori infection

We developed a novel vaccine for Helicobacter pylori based on a poliovirus vector in which capsid genes were replaced with the gene for the B subunit of H. pylori urease (UreB). Mice were vaccinated with UreB or control (L1) replicon and challenged with H. pylori. Twenty percent of mice vaccinated prophylactically with UreB, but 80% vaccinated with L1, and then challenged with H. pylori became infected (P = 0.003). Seventy-three percent of mice with established H. pylori infection vaccinated therapeutically with UreB replicon cleared their infection compared to 33% vaccinated with L1 (P = 0.067). In therapeutically vaccinated mice with residual infection, UreB-vaccinated animals had fewer H. pylori than L1-vaccinated mice (P < 0.05). Anti-urease antibody titres in prophylactically, but not therapeutically, vaccinated mice were markedly higher in animals that received UreB versus L1 replicon (P = 0.01). Vaccination with poliovirus vector containing the gene for the B subunit of H. pylori urease provides significant prophylactic and strong therapeutic protection against H. pylori in mice.     

Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice

The use of Lactococcus lactis as an antigen delivery vehicle for mucosal immunisation has been proposed. To determine whether L. lactis could effectively deliver Helicobacter pylori antigens to the immune system, a recombinant L. lactis expressing H. pylori urease subunit B (UreB) was constructed. Constitutive expression of UreB by a pTREX1 vector resulted in the intracellular accumulation of UreB to approximately 6.25% of soluble cellular protein. Five different oral regimens were used to vaccinate C57BL/6 mice and the immune response measured. One regimen, which consisted of four weekly doses of 10(10) bacteria, followed after an interval of approximately 4 weeks by three successive daily doses, was able to elicit a systemic antibody response to UreB in the mice, although subsequently, a similar regimen produced a significant antibody response in only one out of six mice. The other three regimes, in which mice were vaccinated with two or three sets of three consecutive daily doses of recombinant bacteria over 30 days, failed to elicit significant anti-UreB serum antibody responses. In three regimens, the immunised mice were then challenged by H. pylori strain SS1 and no protective effect was observed. These findings suggest that any adjuvant effects of L. lactis are unlikely to be sufficient to produce an effective immune response and to protect against H. pylori challenge, when used to deliver a weak immunogen, such as UreB.     

Comparison between targeted and untargeted systemic immunizations with adjuvanted urease to cure Helicobacter pylori infection in mice

Outbred OF1 mice infected in a first step with a mouse-adapted Helicobacter pylori strain were immunized in a second step by systemic or mucosal routes: systemic immunizations were performed subcutaneously with adjuvanted urease either in the infra or supra-diaphragmatic region of the body, while mucosal immunization was done with urease in the presence of E. coli heat Labile toxin. Mucosal and subcutaneous immunizations induced in infected mice a significant reduction in bacterial density whatever the site of injection but complete eradication was preferentially observed in mice immunized subcutaneously in the back. Systemic immunization with appropriate schedules and formulations could constitute a valuable approach to cure Helicobacter pylori infection.     

两种非侵入性检测方法对儿童幽门螺杆菌感染的诊断价值分析

目的:研究幽门螺杆菌((Helicobacter pylori,Hp)粪便抗原检测(HpSA)及血清抗幽门螺杆菌试验(Hp-IgG)在诊断儿童HP感染中的价值。方法:以胃黏膜快速尿素酶法(RUT)和组织染色法联合检测结果作为Hp感染诊断金标准,通过对137患儿HpSA及Hp-IgG的检测,并与金标准进行对比分析。结果:137例患儿中金标准阳性75例,阴性62例,以金标准作为Hp感染的诊断标准,HpSA检测的敏感度92.0%,特异度95.1%,准确性93.4%;Hp-IgG检测的敏感度89.3%,特异度91.9%,准确性90.5%。结论:HpSA检测是较理想的非侵入性诊断儿童Hp感染的方法,值得在临床推广。     

苏州市胃肠病患者幽门螺旋杆菌耐药分析

目的 了解江苏省苏州市幽门螺杆菌(H. pylori)对抗生素的耐药情况,为提高幽门螺杆菌感染根除率提供依据.方法 收集经胃镜活检快速尿素酶阳性的慢性胃炎及消化性溃疡患者506例,取胃窦黏膜组织,常规方法培养鉴定幽门螺杆菌,并对分离菌株进行药敏纸片法(KB)鉴定.结果 结果506例慢性胃炎及消化性溃疡患者中,幽门螺杆菌分离率为76.1%;H.pylori对甲硝唑和克拉霉素的耐药率均较高,分别为75.5%及19.4%(P<0.001,P<0.05);对阿莫西林、呋喃唑酮、左氧氟沙星、头孢呋辛、莫西沙星仍保持较高的敏感性,耐药率依次分别为3.6%,2.2%,7.2%,0.0%,5.8%;H. pylori的快速尿素酶试验(RUT)阳性程度越强,培养阳性率就越高.结论 苏州地区H.pylori根除治疗的抗生素应首选阿莫西林,呋喃唑酮、头孢呋辛、左氧氟沙星、莫西沙星等药物,避免使用甲硝唑,慎重选择克拉霉素.     

Serum-derived IgG1-mediated immune exclusion as a mechanism of protection against H. pylori infection

The induction of protective immunity against Helicobacter challenge in a murine model was found to correlate with the magnitude of IgG (serum and gastric lavage) responsiveness to intra-nasal (i.n.) immunisation. IgG1-secreting hybridoma backpacks in Helicobacter pylori (H. pylori)-infected mice revealed serum transudation into the stomach. A Lpp20-specific monoclonal antibody was associated with significantly reduced H. pylori colonisation. Histology revealed aggregates of the remaining H. pylori in these mice, suggesting a role for IgG1-mediated immune exclusion of the bacteria. In vitro immunogold electron microscopy supported this hypothesis, but also suggested that a threshold of H. pylori-specific antibody needs to be maintained if immune exclusion by the host is to overcome immune evasion by the bacteria.     

根除幽门螺杆菌对胃增生性息肉的作用

目的探讨根除幽门螺杆菌（Hp）对胃增生性息肉的作用。方法将胃增生性息肉（息肉直径3～10mm）合并HD感染的48例患者随机分为治疗组及对照组（各24例）。治疗组患者接受四联治疗，包括质子泵抑制剂（奥美拉唑或兰索拉唑）、克拉霉素、枸橼酸铋及替硝唑，疗程2周。对照组仅接受替普瑞酮治疗。受试者在纳入试验前及治疗结束后1～12个月接受定期胃镜检查和Hp测定。结果治疗组和对照组各有22例及21例患者完成整个研究。治疗组有19例（86．4％）Hp得到根除，在用药后1～12个月，平均（6．5±1．1）个月随访中，Hp根除者中15例（78．9茗）胃息肉消失。而在试验开始后l2个月，对照组患者的胃息肉和Hp情况未发生变化（P〈0．01）。结论大多数胃增生性息肉在根除Hp后消失，患者如同时患有胃增生性息肉和Hp感染，在行息肉摘除术前可先尝试Hp根除治疗。     

Effect of diet and Helicobacter pylori infection to the risk of early gastric cancer

BACKGROUND: The association of dietary habits and Helicobacter pylori infection with early gastric cancer is still unclear. METHODS: A hospital-based case-control study was conducted in Korea. Sixty-nine patients were newly diagnosed as having early gastric cancer at the Division of Gastroenterology, Asan Medical Center, and 199 healthy subjects who visited the Health Promotion Center of the this same hospital for annual health examinations were selected as controls. Helicobacter pylori infection status was assayed by ELISA, and information for dietary habits was obtained by interview using a semi-quantitative food frequency questionnaires. Preference for salty taste was also evaluated using a sensitive test. RESULTS: H. pylori seropositivity was observed in 88% of cases, as compared with 75% of controls (OR = 5.3, 95% confidence interval:1.7-16.5). Adaptive salt concentration was significantly and positively associated with early gastric cancer risk (p < 0.01). Decreased risks of early gastric cancer were observed in association with intakes of clear broth, raw vegetables, fruits, fruit or vegetable juices, and soybean curds. On the other hand, a high intake of salt-fermented fish and kimchi were associated with an elevated risk of early gastric cancer. Subjects with positive H. pylori infection and a high salty preference had a 10-fold higher risk of early gastric cancer than subjects without H. pylori infection and with a low salty preference (p for interaction = 0.047). CONCLUSION: Some dietary factors and H. pylori infection are significantly associated with early gastric cancer. In particular, high-salty diets may enhance the effect of H. pyori infection in gastric carcinogenesis.