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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.

Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4(+) but similar numbers of CD8(+) lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4(+) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4(+) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4(+) T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4(+) T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4(+) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.

Helicobacter pylori infection is associated with pronounced infiltration of granulocytes and lymphocytes into the gastric mucosa, resulting in active chronic gastritis that may develop into duodenal ulcer disease or gastric adenocarcinoma. Infiltrating T cells play a major role in the pathology of these diseases, but the signals involved in recruitment of T cells from blood to H. pylori-infected tissues are not well understood. We therefore examined H. pylori-induced T-cell transendothelial migration (TEM). The Transwell system, employing a monolayer of human umbilical vein endothelial cells, was used as a model to study TEM. H. pylori induced a significant T-cell migration, compared to spontaneous migration. CD4+ and CD8+ T cells migrated to the same extent in response to H. pylori, whereas there was significantly larger transmigration of memory T cells compared to naive T cells. Both H. pylori culture filtrate and urease induced migration, and the presence of the H. pylori cag pathogenicity island increased TEM. T-cell TEM was mediated by LFA-1-ICAM-1 interactions in accordance with an increased ICAM-1 expression on the endothelial cells after contact with H. pylori. Migrating T cells had increased expression of activation marker CD69 and chemokine receptors CXCR3, CCR4, and CCR9. Furthermore, T cells migrating in response to H. pylori secreted Th1 but not Th2 cytokines upon stimulation. In conclusion, our data indicate that live H. pylori and its secreted products contribute to T-cell recruitment to the gastric mucosa and that the responding T cells have an activated memory Th1 phenotype.

A Helicobacter pylori-specific in vitro coculture system was established and used to study the role of CD4+CD25+ regulatory T cells (Treg) in gastritis development in mice with H. pylori infection. Effects of therapeutic immunization against H. pylori infection on the Treg function were also studied to better understand the mechanisms leading to postimmunization gastritis in these mice. Depletion of Treg led to extensive proliferation to H. pylori antigens of CD4+ T cells isolated from either naïve, H. pylori-infected or H. pylori-immunized mice. Using the Treg-depleted CD4+ T cells from immunized mice as effector cells, we compared the suppressive efficacy of Treg isolated from naïve, infected or immunized mice and found that Treg from naïve mice, and slightly less efficiently from infected mice, suppressed the CD25- effector T-cell response and in most cases were distinctly more efficacious than Treg isolated from immunized mice. The suppressive efficacy of Treg isolated from the differently treated mice correlated closely with production of interleukin-5 (IL-5) by the Treg and suppression of interferon-gamma and IL-2 production by the CD25- effector T cells. Our study is the first to demonstrate in H. pylori-induced chronic infection, antigen-specific Treg with differential efficacy in suppressing H. pylori proinflammatory T effector cells.

Background and Aims: Helicobacter pylori infection results in an active, chronic inflammation of the gastric mucosa. Previous studies have highlighted the importance of matrix metalloproteinases (MMPs) in diseases involving mucosal inflammation, prompting us to investigate MMP activity in H. pylori-induced gastritis., Methods: Gastric biopsies were obtained from H. pylori-infected and uninfected volunteers, and MMP activity was assessed using substrate gel electrophoresis. MMP production was also evaluated by immunohistochemistry and real time-polymerase chain reaction. In parallel, tissue inhibitors of MMPs (TIMP) levels and TIMP-MMP complexes were examined in corresponding tissues using enzyme-linked immunosorbent assays and Western blotting. Finally, MMP production by gastric macrophages was determined after stimulation with H. pylori., Results: Antral mucosa of H. pylori-infected subjects demonstrated a 19-fold higher MMP-9 activity than that of uninfected individuals. MMP-2 was present at lower levels, but was also increased in H. pylori-infected individuals, while there was no difference in the total levels of TIMP-1 and TIMP-2 between the groups of volunteers. Significant numbers of MMP-9-containing cells were only found in the H. pylori-infected antral mucosa. Tissue-resident macrophages were significantly increased in H. pylori-infected individuals, and double-staining showed MMP-9 colocalized to macrophages. Furthermore, gastric macrophages secreted MMP-9 in response to H. pylori bacteria. A corresponding 10-fold increase of gene expression of MMP-9 was seen in patients infected with H. pylori compared to uninfected individuals., Conclusions: Helicobacter pylori infection results in a substantial increase in MMP-9 and MMP-2 activity in the gastric mucosa, probably contributed to in large part by tissue-resident macrophages, while no changes were seen in the TIMP levels. The net increase in gastric MMP activity is likely to contribute to tissue damage during H. pylori-associated gastritis.

This review deals with the induction and expression of immune responses in the human stomach following mucosal immunisation. As prerequisites for developing a Helicobacter pylori vaccine, the role of gastric inflammation, in particular inflammation induced by H. pylori infection, as well as lymphocyte homing within the common mucosal immune system, and the importance of immunisation route are discussed.

Helicobacter pylori induces symptomatic chronic gastritis in a subpopulation of infected individuals. The mechanism(s) determining the development and severity of pathology leading to symptoms are not fully understood. In a mouse model of H. pylori infection we analysed the influence of immunoregulatory CD4+CD25+ T cells on H. pylori colonization and gastritis. Athymic C57BL/6 nu/nu mice were reconstituted with (a) lymph node (LN) cells (b) LN cells depleted of CD25+ T cells (CD25(-) LN) or (c) not reconstituted at all. Mice were then infected orally with 3 x 10(8)H. pylori SS1 bacteria. At 2 and 6 weeks after the inoculation there was a significant (P < 0.001) reduction in H. pylori colonization in athymic mice transferred with CD25(-) LN cells compared to mice transferred with LN cells. Colonization was still reduced at 12 weeks after inoculation. Mice transferred with CD25(-) LN cells showed an earlier onset and increased severity of gastritis as compared to mice receiving LN cells. Splenic cells isolated from mice receiving CD25(-) LN cells produced the highest level of IFN-gamma on stimulation with H. pylori antigens in vitro, had a higher H. pylori-specific DTH response and increased infiltration of CD4+ T cells and macrophages in the gastric mucosa. Athymic mice not transferred with T cells had persistent high H. pylori colonization and displayed a normal gastric epithelium without inflammatory cells. In conclusion, CD4+CD25+ cells reduce immunopathology in H. pylori infection, possibly by reducing the activation of IFN-gamma producing CD4+ T cells, even at the expense of a higher H. pylori load in the gastric mucosa.

Helicobacter pylori colonize the human stomach and duodenum. The infection has been shown to induce a strong T-cell response in the stomach, whereas the response within the duodenum has been poorly characterized. Furthermore, it remains to be elucidated whether the T-cell response may contribute to ulcer formation in the host. In this study, the frequency of different T-cell subsets, their degree of activation and expression of co-stimulatory receptors in biopsies from the duodenum as well as the antrum were studied by immunohistochemistry and flow cytometry. It was also evaluated whether there are differences in the T-cell responses between duodenal ulcer patients and asymptomatic carriers that might explain why only 10-15% of the infected subjects develop duodenal ulcers. The frequencies of CD4+, CD8+ and CD45RO+, i.e. memory T-cells, were significantly increased in the antrum, and the number of CD25+ cells was considerably higher in both the antrum and duodenum of duodenal ulcer patients and asymptomatic carriers as compared to uninfected individuals. Interestingly, the levels of immunosuppressive CTLA-4+ cells were significantly higher in the duodenum of duodenal ulcer patients, as compared to the asymptomatic carriers. H. pylori cause activation of T-cells in the duodenum as well as in the stomach. Our observation of higher levels of CTLA-4+ cells in the duodenum of duodenal ulcer patients than in the asymptomatic carriers suggests that a suppressive T-cell response may be related to the development of duodenal ulcers.

Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25(high) T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25(high) regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25(high) T cells suppressed H. pylori-induced responses in cocultures with CD25(low/-) cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.

The aim of this study was to recombinantly produce and purify Helicobacter pylori adhesin A (HpaA) from Escherichia coli and compare it to purified native H. pylori HpaA, for potential use as a vaccine antigen. The hpaA gene was cloned from H. pylori, transferred to two different expression vectors, and transformed into E. coli. Expression of rHpaA was analysed by immunoblot, inhibition ELISA, and semi-quantitative dot-blot. Using affinity chromatography, rHpaA was purified from E. coli and native HpaA from H. pylori. The binding of both purified proteins to sialic acid was analysed and antibody titres to native and rHpaA were compared after intraperitoneal immunisation of C57/Bl mice. The rHpaA protein was highly expressed in E. coli from both vectors. Purified recombinant and native HpaA bound similarly to fetuin but also to the non-sialylated asialofetuin. Both native HpaA and rHpaA induced comparable amounts of specific antibodies in serum after immunisation and they were identical in double immunodiffusion. In conclusion, rHpaA was successfully produced in E. coli. Purified rHpaA showed biological properties similar to those of native HpaA isolated from H. pylori and may therefore be further used as an antigen in the development of a vaccine against H. pylori infection.

The expression of the virulence-associated genes ureA, encoding the urease subunit A, and nap, encoding the neutrophil activating protein, in Helicobacter pylori grown both in the stomach of C57/Bl6 mice and in Brucella broth was quantified by quantitative competitive reverse transcriptase-PCR using a homologous RNA standard (competitor) and an external standard (16S rRNA). The results showed that the ureA and nap transcripts were increased up to 15 and 80 times, respectively, in vivo compared to in vitro. The transcription of ureA and nap also differed in that ureA showed highest expression early in infection in mice whereas nap transcription was variable throughout the 18-week infection period.

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath., (Copyright 2001 Academic Press.)

Background: In a previous study, we found that oral vaccination induces strong B cell responses in the stomach of Helicobacter pylori infected but not of uninfected individuals. In this study, we have evaluated the possibility of inducing gastric immune responses in H pylori infected volunteers by intestinal and gastric immunisation., Methods: H pylori infected subjects were given two doses of an inactivated cholera vaccine, either intestinally via an endoscope approximately 30 cm distal to the pylorus sphincter or intragastrically as small droplets applied directly onto the stomach mucosa. Uninfected individuals received the vaccine by standard oral procedure. Vaccine specific antibody secreting cells in antral and duodenal biopsies were detected by the enzyme linked immunospot assay technique before and seven days after the second immunisation., Results: Intestinal immunisations resulted in induction of vaccine specific gastric IgA secreting cells in five of eight volunteers. This immunisation schedule also gave rise to specific duodenal antibody secreting cells in seven of eight individuals. Local gastric immunisation resulted in the induction of specific B cells in the gastric mucosa of four of eight volunteers. Gastric antigen application also resulted in B cell responses in the duodenum in all volunteers. Uninfected volunteers receiving the vaccine perorally responded in the duodenum but not in the stomach., Conclusions: H pylori infection increases the ability of the gastric mucosa to serve as an expression site for intestinally induced B cell responses. These findings are of importance when designing a therapeutic H pylori vaccine, and based on our results such a vaccine can be delivered along the whole upper gastrointestinal tract.

Helicobacter pylori infection can cause duodenal ulcers and may also induce gastric adenocarcinoma. The bacteria colonize the gastric mucosa and areas of gastric metaplasia in the duodenum for decades, resulting in active chronic inflammation in the infected areas. A characteristic feature of the infection is the ongoing recruitment of neutrophils to the infected sites. To evaluate the role of H. pylori lipopolysaccharides (LPS) in the recruitment of leukocytes to the gastric mucosa, we have examined the cytokine and chemokine production from human monocytes stimulated with LPS isolated from different H. pylori strains, as well as from several other gram-negative bacteria. Our results show that H. pylori LPS induce a large production of neutrophil-recruiting CXC chemokines (interleukin-8 and growth-related oncogene alpha) from purified human monocytes, to almost the same extent as Escherichia coli LPS. However, and in agreement with previous studies, H. pylori LPS was much less potent in inducing production of proinflammatory cytokines by purified human monocytes and was also a weak inducer of the CC chemokine RANTES. There was no difference between LPS preparations from different H. pylori strains in their ability to induce cytokines and chemokines. The preferential production of CXC chemokines after stimulation with H. pylori LPS indicates an important contribution of this molecule in maintaining neutrophil recruitment during the infection, irrespective of the infecting strain.

Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori.

The 26 kDa protein of Helicobacter pylori, with 67% amino acid identity to alkyl hydroperoxide reductase (AhpC) of Campylobacter jejuni, was studied. We wanted to evaluate it the protein has AhpC activity. Therefore, an Escherichia coli mutant defective for alkyl hydroperoxide reductase and a plasmid expressing the 26 kDa protein from H. pylori were used in complementation studies. The complemented E. coli mutant showed a decreased sensitivity to cumene hydroperoxide indicating that the 26 kDa protein of H. pylori has AhpC activity and could be of importance in the defence against oxidative stress. Furthermore, Northern blot analysis detected one mRNA transcript of approximately 700 bp which is in agreement with the gene being transcribed as a single gene with its own promoter. This promoter region was further characterized by primer extension experiments. Additional studies on how environmental factors, such as long term growth and pH, can affect the transcription of the gene were performed on two H. pylori strains. We found that low pH and long term growth repressed transcription of the gene. Attempts to construct a mutant deficient for the gene in H. pylori were unsuccessful., (Copyright 2000 Academic Press.)

Background: We have investigated the possibility that the same patients may be colonized by Helicobacter pylori strains of different genotypes or phenotypes in the antrum as compared to in the duodenum. The strains were typed for DNA fingerprints, different lipopolysaccharides (LPS), and Lewis antigen expression on the O-side chains of LPS., Materials and Methods: Polymerase chain reaction (PCR) amplifications using primer sequences (i.e., the Enterobacterial Repetitive Intergenic Consensus [ERIC]) and randomly amplified polymorphic DNA (RAPD) elements were performed to asses chromosomal DNA diversity between H. pylori strains. The expression of different LPS types and Lewis antigens in the various H. pylori isolates were determined by whole bacterial enzyme-linked immunosorbent assays using monoclonal antibodies., Results: Duodenal ulcer patients had different H. pylori genotypes in the duodenum as compared to in the antrum as shown by ERIC-PCR (44%) and by RAPD-PCR (75%). Different DNA patterns were found among the strains that were isolated from various regions of the duodenum in 4 of 16 patients (25%) as shown by ERIC-PCR and in 8 of 16 patients (50%) as shown by RAPD-PCR. Sixty-three percent of the duodenal ulcer patients had H. pylori strains with a different Lewis antigen phenotype in the duodenum as compared to in the antrum, and 3 of 16 patients (19%) had strains with different Lewis antigens expressed by strains from different duodenal biopsies from the same patient., Conclusion: The results suggest that a mixed population of different H. pylori strains with marked variation, both genotypically and phenotypically, colonize the same patient.

The discovery of the gastric pathogen Helicobacter pylori has created a need for accurate methods to study immune responses locally in the human stomach. Therefore, we have developed a quick and easy method for extraction of antibodies from gastric biopsies using saponin and compared this method with the more laborious analysis of antibody-secreting cells (ASCs) from gastric biopsies. We have also analyzed the antibody content in gastric aspirates, saliva and plasma. There was a strong correlation between the total IgA levels in the biopsy extracts and the frequencies of IgA-secreting cells. In addition, the IgA and IgG levels against a H. pylori whole membrane preparation and purified urease in the biopsy extracts correlated well with the frequencies of specific IgA and IgG secreting cells. However, the antibody levels in gastric aspirates, saliva and plasma specimens did not correlate with the frequencies of corresponding ASC in the gastric biopsies. Thus, the saponin extraction method is suitable for monitoring local antibody responses in the stomach, while analyses of gastric aspirates, saliva or plasma are not appropriate for this purpose.

The host immune response to Helicobacter pylori infection might be of importance with regard to the outcome of infection by this organism, e.g., to explain why only a proportion of infected subjects develop peptic ulcers. In this study we have analyzed the local response of different cytokines-i.e., the proinflammatory interleukin-1beta, (IL-1beta), IL-6, tumor necrosis factor alpha, and IL-8; the immunoregulatory gamma interferon (IFN-gamma); and IL-4; and the anti-inflammatory transforming growth factor beta (TGF-beta)-in antral biopsy specimens from H. pylori-infected duodenal ulcer (DU) patients and asymptomatic (AS) carriers (i.e., with chronic gastritis only). For comparison, biopsy specimens from uninfected healthy individuals were also analyzed. An immunohistochemical technique was used to allow quantification of the cytokine responses as well as identification of the cell types associated with the cytokine expression. We found that the levels of all of the studied cytokines except IL-4 were increased in the H. pylori-infected subjects compared to the levels in the healthy individuals. Our results indicate that the antral cytokine response is of the Th1 type since IFN-gamma, but not IL-4, was up-regulated both in H. pylori-infected DU patients and in AS carriers. However, there were no significant differences in either proinflammatory or immunoregulatory cytokine levels when H. pylori-infected subjects with and without peptic ulcers were compared. Some of the cytokines, particularly IL-1beta and TGF-beta, were also found in the gastric mucosae of healthy, uninfected subjects. We also showed that the gastric epithelium contributes substantially to the antral cytokine response of the proinflammatory cytokines IL-1beta and IL-6 in addition to IL-8.

We have evaluated the possibility of inducing antibody responses locally in the human stomach as a prerequisite for the development of a vaccine against Helicobacter pylori. Both H. pylori-infected and noninfected subjects were immunized with an oral B subunit whole cell (BS-WC) cholera vaccine, and total and vaccine-specific antibody-secreting cells (ASC) were determined by the enzyme-linked immunospot (ELISPOT) technique in cells isolated from the antrum and duodenum, respectively, before and after vaccination. Most of the subjects responded to the vaccination with high frequencies of vaccine-specific ASCs in the duodenum as well as high-serum antibody titers, and no significant differences were seen in the responses between H. pylori- infected and noninfected subjects. When studying the gastric mucosa, on the other hand, there were dramatic differences between the H. pylori-infected and the noninfected subjects. Thus, whereas none of the noninfected subjects responded to the immunization in antrum, most of the H. pylori-infected subjects had high frequencies of vaccine-specific ASCs in this location after vaccination. Furthermore, the H. pylori-infected subjects had strikingly higher (as a mean 80-fold) frequencies of total IgA-secreting cells in antrum than the noninfected subjects, whereas the frequencies of total IgA-secreting cells in the duodenum were comparable between the groups. In conclusion, these results demonstrate the possibility of inducing antibody responses locally in the gastric mucosa of H. pylori-infected individuals, a finding with obvious implications for the future development of a therapeutic vaccine against H. pylori.

In this study we have determined systemic and local antibody responses against different Helicobacter pylori antigens in H. pylori-infected and noninfected subjects. In addition, we studied whether differences in antibody responses between patients with duodenal ulcers and asymptomatic H. pylori carriers might explain the different outcomes of infection. Sera and in most instances gastric aspirates were collected from 19 duodenal ulcer patients, 15 asymptomatic H. pylori carriers, and 20 noninfected subjects and assayed for specific antibodies against different H. pylori antigens, i.e., whole membrane proteins (MP), lipopolysaccharides, flagellin, urease, the neuraminyllactose binding hemagglutinin HpaA, and a 26-kDa protein, by enzyme-linked immunosorbent assay. The H. pylori-infected subjects had significantly higher antibody titers against MP, flagellin, and urease in both sera and gastric aspirates compared with the noninfected subjects. Furthermore, the antibody titers against HpaA were significantly elevated in sera but not in gastric aspirates from the infected subjects. However, no differences in antibody titers against any of the tested antigens could be detected between the duodenal ulcer patients and the asymptomatic H. pylori carriers, either in sera or in gastric aspirates.

In this study, we raised monoclonal antibodies (MAbs) against three conserved Helicobacter pylori antigens, i.e., the N-acetylneuraminyllactose-binding fibrillar hemagglutinin, HpaA; the flagellin subunits, FlaA and FlaB; and a species-specific 26-kDa protein. The MAbs were used for the development of sensitive inhibition enzyme-linked immunosorbent assays for quantification of these antigens in H. pylori during various culture conditions. The quantities of these antigens varied considerably (up to 8-fold) during different culture procedures and between strains (up to 10-fold).

The capacity of Helicobacter pylori to induce strain specific immune responses was studied in adult Swedish volunteers. Sera and gastric aspirates from 11 H. pylori-infected subjects were tested for specific antibody levels against, respectively, lipopolysaccharides (LPS) and total membrane preparations (MPs) prepared from the study subjects' own strains, as well as with corresponding antigens from two reference H. pylori strains or heterologous strains collected from other subjects within the study. It was found that sera from five of the 11 subjects had significantly higher IgA antibody titres against LPS from the homologous strain than against LPS from either of the reference strains and in five cases sera reacted with higher IgG titres against the homologous LPS than with LPS preparations from the reference or heterologous patient strains. Analyses of specific titres against MPs revealed that six sera had higher IgA titres and four sera had higher IgG titres against MPs prepared from the subjects' own strains than against MPs from either of the two reference strains. Determination of specific antibodies in gastric aspirates revealed significantly higher IgA titres against LPS from the homologous H. pylori isolate than against LPS from the two reference strains in five cases, and six aspirates reacted in higher IgA titre with the homologous H. pylori MPs. Results from immunoblotting analyses of sera support induction of strain specific immune responses against H. pylori LPS. By means of specific monoclonal antibodies against H. pylori LPS, antigenic heterogeneity between the different LPS preparations tested was confirmed., (Copyright 1997 Academic Press Limited.)

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.