* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Helicobacter pylori vaccines and mechanisms of effective
Sociality and disease transmission wikipedia , lookup
Childhood immunizations in the United States wikipedia , lookup
Herd immunity wikipedia , lookup
Molecular mimicry wikipedia , lookup
Immune system wikipedia , lookup
Hygiene hypothesis wikipedia , lookup
DNA vaccination wikipedia , lookup
Social immunity wikipedia , lookup
Vaccination wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Neonatal infection wikipedia , lookup
Human cytomegalovirus wikipedia , lookup
Immunocontraception wikipedia , lookup
Adaptive immune system wikipedia , lookup
Hospital-acquired infection wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Hepatitis B wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Infection control wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Immunology and Cell Biology (2001) 79, 67–73 Theoretical Article Helicobacter pylori vaccines and mechanisms of effective immunity: Is mucus the key? P H I L I P S U T TO N School of Microbiology and Immunology, University of New South Wales, Sydney, New South Wales, Australia Summary In this theoretical article, the hypothesis is proposed that immunization against gastric helicobacter infection is mediated by CD4+ T-cell induced changes in mucus production. Vaccine development for the gastric pathogen Helicobacter pylori has encountered several problems. Resolving these problems is impeded by our lack of understanding of the mechanisms by which the immune response influences bacterial colonization. Protective immunity requires CD4+ T cells, but the majority of helicobacters are located in the mucus of the gastric lumen, away from the epithelial surface. Evidence suggests that this mechanism functions independently of antibodies, so how this is achieved is unknown. Clues to this mechanism may be provided by immune clearance of nematode infection. Similar to H. pylori, expulsion of the intestinal nematode, Nippostrongylus brasiliensis, in rodents is mediated by CD4+ T-cell changes in the numbers of goblet cells and the type of mucins secreted into the gut. Immune-mediated changes in secretion of gastric mucins could similarly be responsible for the reductions in helicobacter colonization seen in immunized animals. Helicobacter pylori are highly motile bacteria that have evolved to inhabit their specialized niche. Alterations in their mucus environment could influence their motility, such that the bacteria cannot remain efficiently within the mucus and are flushed away. Key words: effector mechanism, Helicobacter pylori, immunisation, mucus. Introduction Over the last decade, there has been significant effort focused upon the development of a vaccine against the human gastric pathogen, Helicobacter pylori. This bacterium was discovered 18 years ago in Perth, Australia, in the stomachs of individuals exhibiting chronic gastritis.1,2 Following initial scepticism, it has now been conclusively demonstrated that H. pylori is a key aetiological factor in the majority of peptic ulcers3 and is listed as a class I carcinogen due to its association with the development of gastric adenocarcinoma and mucosa associated lymphoid tissue (MALT) lymphoma.4–6 Although antimicrobial therapy is available, the cost and the emerging drug resistance7 mean that an effective vaccine would be of great benefit, particularly in the poorer developing countries, which have the highest incidence of gastric cancer. Unfortunately, some issues regarding the final vaccine remain to be resolved; a situation made more complicated by our lack of understanding regarding the effector mechanisms induced by immunization. This theoretical article discusses the evidence that may explain the immune events that influence colonization by gastric helicobacters. Helicobacters and vaccines The first animal model developed to investigate helicobacters in gastric pathology used a mouse infected with Helicobacter Correspondence: P Sutton, School of Microbiology and Immunology, University of New South Wales, Sydney, NSW 2052, Australia. Email: [email protected] Received 15 June 2000; accepted 21 August 2000. felis, a very close relative of H. pylori, which was isolated from a cat.8 Helicobacter felis was found to not only colonize mouse gastric tissue in abundance, but also to induce severe gastritis. Mouse-colonizing strains of H. pylori are now available,9,10 although they do not induce a severe inflammatory response in mice, which make them rather poor models for studying helicobacter-induced gastritis. They do, however, provide an ideal model for investigating protective immunity against H. pylori infection. The use of these helicobacter–animal models for immunization studies has raised a number of important issues. First, although it was initially believed that vaccination induced sterilizing immunity, which completely prevented or cleared infection, it has subsequently been realized following the development of more sensitive assays that although bacterial colonization is greatly reduced, some bacteria always remain.11,12 The second issue is the requirement for a mucosal adjuvant, such as cholera toxin (CT) or heat labile toxin from Escherichia coli (LT), for effective immunity. Unfortunately humans are more sensitive to these reagents than are rodents, so they are considered too toxic for use in a final human vaccine. Currently there is no mucosal adjuvant that is suitable for use in humans. Some progress has been made with the use of detoxified forms of these adjuvants13 and with live vectors, such as recombinant attenuated salmonella expressing helicobacter antigen.14–17 The third potential problem involves a phenomenon known as postimmunization gastritis. Prophylactic immunization of mice with subsequent challenge with helicobacters can induce a more severe gastritis than caused by infection alone.11,18,19 68 P Sutton Given that it is the inflammation that leads to the associated diseases, this would not be a desirable outcome. Postimmunization gastritis appears to be driven by the residual bacteria that are not cleared by immunization, because treatment of the immunized challenged mice with antimicrobials to remove the remaining H. felis prevented the exacerbation of gastritis.11 Thus, in order to create a human vaccine it may be necessary to improve immunization to produce sterilizing immunity, circumvent the adjuvant problem and ensure that the immunizations will not, in fact, increase the likelihood of an individual developing helicobacter-induced pathologies. All of these are difficult to achieve without an understanding of the effective immune mechanisms, induced by vaccination, that influence colonization by H. pylori. Effective immunity against H. pylori infection: What is known? In humans, infection with H. pylori normally occurs during childhood and, if left untreated, will generally remain in the gastric lumen for the lifetime of the individual. A small proportion of these organisms adhere to the luminal surface of the gastric epithelium, but the majority of the colonizing bacteria are ‘free-swimming.’ The presence of these bacteria induces a chronic gastritis, which lasts for decades. This inflammation is a cell-mediated response driven by Th1-type cytokines, as shown by numerous studies using both infected humans and animal models.20,21 Despite this extremely longterm immune response, the infection persists. Antibodies Given the location of the infection in the gastric lumen, it was initially expected that antibodies would mediate protective immunity. Even before animal models became available, Czinn and Nedrud found that immunization of mice and ferrets with killed H. pylori and CT produced a significant intestinal and serum antibody response.22 When the H. felis mouse model was developed and protective immunity demonstrated,23 it was found that oral delivery of helicobacterspecific IgA monoclonal antibodies at the same time as challenge with H. felis protected mice against colonization.24 Many of the following studies found an association between protective immunity and serum or secretory antibody responses.19,25,26 It was an early paper from 1986 by Wyatt et al., who looked at tissue from individuals infected with Campylobacter pyloridis (now H. pylori), which provided the first suggestion that antibodies may not be effective.27 Bacteria colonizing the gastric tissue of patients with gastritis were coated with IgA.27 They also found that the bacteria infecting patients with active gastritis (a form with neutrophil infiltration, which is associated with greater progression to helicobacter-induced diseases) were frequently coated with IgG and IgM.27 The antibody response in these individuals was clearly having no success in clearing the infection. Doubts regarding the role of antibodies were further raised by several immunization studies that found no association, or even an inverse correlation, between protective immunity and antibody production.20,28 Most convincingly, immunization of antibody-deficient mice produces the same level of protection against both H. felis and H. pylori as is present in wild-type mice.29–31 Thus, normal protection can occur even in the complete absence of antibodies. This demonstrates that antibody independent immune mechanisms exist that are induced by immunization and can influence the colonization of bacteria that inhabit the gastric lumen. T cells In contrast, there is clear evidence that T lymphocytes are critically required for protective immunity against helicobacter infection. Experiments using major histocompatibility complex (MHC)-deficient mice have demonstrated that CD4+, but not CD8+ T cells, are essential for effective immunity against H. pylori in mice. Immunization of mice deficient in MHC class I molecules produces the same protective immunity as wild-type mice. However, if mice are deficient in MHC class II molecules, used by the immune system to present antigen to CD4+ Th cells, then no protection against bacterial colonization results.29,32 It remains uncertain whether it is the Th1- or Th2-type response that is important. Some evidence suggests a role for Th2-type immunity. Adoptive transfer of helicobacter-specific Th1 cell lines has no effect on bacterial colonization after challenge, but transfer of helicobacter-specific Th2 cell lines induces a significant reduction in infection levels.33 Additionally, protection following immunization is associated with increased IL-4 and decreased IFN-γ secretion by splenic and gastric lymphocytes.20,34 Gastric infection with helicobacters naturally produces an ineffective Th1 inflammatory response,20,21 so it came as no surprise that Th2 cells were found to be associated with protection. However, evidence is accumulating that questions this role for Th2 immunity. Immunizations on IL-4-deficient mice have found that although protective efficacy is reduced, it is not lost, suggesting that some protection is possible in the absence of Th2 immunity.35 In contrast, several studies have suggested that protection is lost in mice deficient in IFN-γ or its receptor.36,37 In addition, a reduction in H. felis colonization, induced by adenovirus co-infection, is dependent on IFN-γ and IL-12.38 Whether it eventuates to be Th1, Th2 or both CD4+ cell types that induce protective immunity, it is likely that they only constitute the trigger of the immune response. With the demonstration that immune mechanisms exist that can function independently of antibodies, it is necessary then to speculate on alternative effector mechanisms that can prevent or inhibit helicobacter colonization of the gastric mucosa. To do this we need to consider what is known regarding the interaction of the bacterium and its habitat. Helicobacter and the gastric mucosa Helicobacter pylori inhabits the gastric mucosa and is mainly found located free in the mucus layer, with some binding to the epithelial surface both in infected humans39,40 and in mice.10 Figure 1 shows a gastric mucus scraping taken from a person infected with H. pylori and the spiral bacteria are clearly present in large numbers in the mucus. Only a low proportion of colonizing H. pylori actually adhere to the epithelium.39,41 Schreiber et al., using a microinjection system, have analysed the location of H. felis within the mucus of infected Immunity and H. pylori: A role for mucus? 69 Figure 1 Light micrograph showing a gastric mucus scraping from an individual infected with Helicobacter pylori. mouse stomachs: three-quarters were found between 5 and 20 µm from the epithelial surface and no bacteria were located closer than 5 µm.42 Thus, H. felis does not appear to adhere at all to the epithelium. Despite this, effective immunity against infection with H. felis induced by immunization appears to parallel that obtained with the H. pylori mouse model. Effects of H. pylori infection on gastric mucus production Mucus is a viscous gel secreted by mucosal cells lining the gastrointestinal tract, including goblet cells and epithelial cells. The secreted mucus forms a protective barrier at the mucosal surface and its main components are heavily glycosylated proteins called mucins. The type of mucin produced and the way in which it is glycosylated varies between different sections of the gastrointestinal tract, reflecting the different demands required of the mucus at individual sites. The luminal surface of the stomach is lined with gastric columnal epithelial cells, which secrete neutral mucopolysaccharides. There is much evidence to suggest that H. pylori changes mucus production by the host. Before the discovery of H. pylori, it was noted that individuals with gastritis or peptic ulcer disease exhibited changes in mucin production at the gastric surface.43 There then followed many studies that proposed that these changes may be due to infection with H. pylori, although most of these were performed in vitro. Micots et al. found that culture of H. pylori with a human mucus secreting cell line strongly inhibited mucin secretion induced by acute stimulation with secretagogue agents.44 They concluded that H. pylori can directly impair the secretory functions of mucous cells. Supporting this, it has been found, using cultured gastric epithelial cells in vitro, that H. pylori lysate inhibits mucus production.45 Slomiany et al. have found that H. pylori produces active enzymes, both proteases and lipases, that degrade gastric mucins isolated from healthy individuals, in vitro.46,47 However, the significance of this has been contended by Markesich et al., who have examined the mucus from patients infected with H. pylori before and after bacterial eradication. In this in vivo study, they found that the gastric mucus was actually more viscous after the loss of H. pylori infection than the mucus obtained from the same individual while still infected.48 Slomiany et al., themselves later supported the Markesich et al. study, in a paper where they demonstrated that the physiological and mechanical protective properties of mucus were restored after H. pylori eradication.49 Adhesion between H. pylori and gastric mucins Numerous studies have investigated the binding properties of H. pylori. In vitro experiments have provided evidence that the adhesion of H. pylori to epithelial cell lines50,51 and to mucins52–54 may involve sialic acid and sulfated oligosaccharides. This has been supported in vivo by Genta et al., who have found that the adhesion of H. pylori to areas of intestinal metaplasia is associated with the expression of sulfomucins on the gastric tissue.55 Another bacterium that can infect the mucosal surface is Pseudomonas aeroginosa, an important pathogen in cystic fibrosis. This bacterium infects the lungs and expresses mucinbinding proteins with which it adheres to bronchial mucins. This binding is inversely correlated with iron availability, so in conditions of low iron, mucin-binding protein expression and thus mucin binding increase.56 One unusual feature about H. pylori, which was revealed upon genomic sequencing of two strains, is the high number of iron-scavenging molecules 70 P Sutton it produces.57,58 This observation suggests that iron performs an important role for H. pylori, and it is tempting to speculate that this is perhaps related to mucin-interactions. A lesson from a nematode? Nippostrongylus brasiliensis is a nematode which inhabits the mucus of the jejunum of rats. Like H. pylori, the organism is topographically distant from immune cells. However, this infection does induce a protective immune response. So can we gain clues regarding immunity against H. pylori, by looking at what is known about N. brasiliensis? The theory on how the immune system of rats can induce expulsion of N. brasiliensis is intriguing. The current hypothesis, first proposed by Ogilvie and Love, is that this expulsion is a two-step process, whereby specific antibodies cause damage to the worm and a subsequent lymphocytemediated non-specific response leads to worm expulsion.59 Evidence to support this hypothesis comes from the use of ‘damaged’ worms; that is, worms that are in the process of being expelled. If damaged worms are used to infect naïve rats, then the worms are expelled in an accelerated manner.60 Evidence suggests that this latter non-specific step is caused by a T-cell mediated alteration in goblet cell numbers and mucin production. Infection of immunocompetent rats with larvae of N. brasiliensis produces an acute infection, which the host begins to clear after 10 days, with most worms expelled by 14 days postinfection.61 If purified immune T cells from rats infected with N. brasiliensis for 10 days are adoptively transferred into naïve rats, which are challenged at the same time with N. brasiliensis, the response to the nematode is greatly accelerated. In rats receiving these specific T cells, clearance begins after day six, with all worms completely expelled by day eight.61,62 As early as 1963, it was noted that goblet cells in the intestinal mucosa of rats increased in number just prior to and during expulsion of N. brasiliensis.63 It was later found that a similar increase in goblet cell number was induced on transfer of purified immune T cells from 10 day N. brasiliensis infected rats into newly infected recipient rats, but not when these T cells were transferred into uninfected controls.62 Histochemical staining revealed that during expulsion of the nematode by a rat, there was a change in the composition of goblet cell mucins.64 By the use of lectin immunostaining, Oinuma et al. have demonstrated that at the time of expulsion, goblet cell mucins begin to strongly express a variety of glycoconjugates, including sialic acid, N-acetyl-Dgalactosamine (GalNAc) and N-acetyl-D-glucosamine.65 When nude rats, which lack normal T cells, were infected with N. brasiliensis, the worms were not cleared and there was no increase in either goblet cell numbers or the type of mucins produced.60 The same authors, using a mouse model, have found that spontaneous cure of N. brasiliensis infection is inhibited by treatment of mice with antibodies against the CD4, but not CD8 T cell marker.66 Most significantly, they have found that the in vivo depletion of CD4+ cells reduces the secretion of intestinal mucus, which is associated with the loss of worm expulsion. There are at least two hypotheses explaining how the two-step model may work. The first proposes that a fully functional N. brasiliensis in some way inhibits the mucinaltering activity of the T cells. Thus, in the first step, an antibody mediated attack occurs by which the worm is damaged and prevents the production of the inhibitory signal, leaving the T cells free to induce expulsion. The second hypothesis states that the damage inflicted on the worm causes release of a stimulatory signal that leads T cells to induce worm expulsion. Why changes in host mucin production lead to expulsion of a reasonably complex organism, such as a nematode worm, is also unknown, but has some degree of specificity. When rats were co-infected with N. brasiliensis and the related nematode Strongyloides ratti, the worms were expelled at different times.67 Expulsion of N. brasiliensis correlated with changes in mucus, whereas loss of S. ratti infection occurred on development of a significant mast cell response. The authors have proposed that there is an interaction between the mucins and a specific function of N. brasiliensis.67 Bringing it all together: The conclusion Helicobacter pylori is highly evolved to survive and indeed thrive in a specialized ecological niche, which is uninhabitable to almost all other infectious organisms. This environment in which H. pylori exists is one of low pH, such that even the gastric tissue protects itself by secretion of a thick mucus layer. Part of the evolution of H. pylori into this niche involves the utilization of this mucus, at least in part for protection against the harsh environment of the stomach. The majority of H. pylori are localized in the mucus away from the epithelial surface and, in the case of H. felis colonization of mice, no bacteria adhere to the gastric tissue. These bacteria not only directly interact with mucus and mucin proteins, but infection can also alter their synthesis. One possible interpretation is that the host is responding to limit infection by altering the mucus in an attempt to flush out the bacteria. Alternatively, perhaps it is part of the strategy of the bacteria, to favour its colonization and in fact reduce its expulsion. Given the lifelong infection that is the normal result, the latter is perhaps the most likely explanation. Using animal models of helicobacter infection, it has been shown that immunization can induce an effective immune response to often quite dramatically reduce bacterial colonization. The mechanism of this process is unknown, but involves CD4+ T cells and is antibody independent. Despite the finding that there is a clear distance between the helicobacters and the epithelial surface, effective immunity is still achievable. It is unlikely that a direct cell action can be responsible for influencing colonization. There are many similarities between the situation of protective immunity against helicobacter infection and the clearance of N. brasiliensis infection by rodents. Both organisms live in gastrointestinal mucus, predominantly distal from the epithelial surface, and clearance is mediated by a process involving CD4+ T cells. It is interesting that the main association of worm expulsion and host immunity is the CD4+ T-cell driven alteration in mucus production. Considering these facts, the hypothesis is presented here that immunization against gastric helicobacter infection induces CD4+ T-cell mediated changes in mucus production. If true, how could changes in mucus lead to expulsion of Immunity and H. pylori: A role for mucus? Figure 2 71 Electron micrographs of gastric mucus scrapings from mice showing (a) Helicobacter pylori and (b) Helicobacter felis. the bacteria? There are several possibilities. Perhaps a change in mucus consistency could produce greater exposure of the helicobacters to the stomach’s acid secretions. This is supported by our knowledge that H. pylori is sensitive to environmental pH.68 It is more likely, however, that the environmental change is such that the bacteria cannot remain efficiently within the mucus and are flushed away. Gastric helicobacters are very motile organisms, possessing flagella for propulsion and a spiral morphology (shown in Fig. 2), which allows rapid corkscrew movement through their viscous surroundings. Hazell et al. have examined the movement of H. pylori in various solutions of methyl cellulose and have found that in a highly viscous environment the bacteria has amazing motility, moving up to 78 µm/s.40 If for a moment we imagined H. pylori as 2 m long, this would equate to a speed of approximately 90 km/h, while travelling through a dense gel. When the viscosity of the environment is increased or decreased, the velocity of the spiral helicobacter rapidly declines.40 The mucus layer of the stomach is a dynamic habitat, with a constant flow of mucus and other gastric secretions down the alimentary tract. It is not unreasonable to assume that the motility of gastric helicobacters is essential for them to remain in the gastric mucosa and that impediment of this motility by changes in mucus viscosity could well lead to rapid expulsion. Motility impediment could possibly result from either an increase or a decrease in viscosity. It is also possible, though less likely, that the altered mucins bind to the flagella, producing the same result. In a paper written over 20 years ago, and 5 years before the discovery of H. pylori, Nawa et al.62 wrote: ‘Even though N. brasiliensis causes severe pathological changes in the intestine, the parasites remain on the surface of the mucosa and do not penetrate the epithelium. This observation raises important questions as to how lymphoid cells can exert a direct effect on the parasites. Most workers agree that a specific antibody has an important role, although it is generally thought that the final phase of expulsion is brought about by nonspecific mediators.’ It appears that helicobacter researchers today are asking the same questions that have been asked for over two decades by immunologists studying nematode clearance. We are now in a position to use this knowledge obtained from nematodes to examine and unravel a mystery of immune function and H. pylori infection. Solving this mystery, to explain this poorly recognized and understood mucosal immune function, will be invaluable in vaccine design for protection against H. pylori. Current development is occurring very much in the dark, with improvements difficult in the face of our ignorance regarding the effector mechanisms. Beyond that, understanding this mechanism will have great implications in the development of immune strategies for combatting other lumenal dwelling pathogens. Acknowledgements I would like to gratefully acknowledge the significant contribution of Professor Chris Parish, who brought to my attention the concept that T cells can mediate effective immunity by alterations in mucus production. My appreciation also goes to Jani O’Rourke for the photo and electron micrographs of the bacteria. References 1 Marshall BJ. Unidentified curved bacillus on gastric epithelium in active chronic gastritis. Lancet 1983; 1: 1273–5. 2 Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984; 1: 1311–5. 3 Bell GD, Bate CM, Axon ATR et al. Symptomatic and endoscopic duodenal ulcer relapse rates 12 months following Helicobacter pylori eradication treatment with omeprazole and amoxycillin with or without metronidazole. Aliment. Pharmacol. Therapeut. 1996; 10: 637–44. 4 International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon: World Health Organisation, 1994; 177–240. 72 P Sutton 5 Correa P, Miller M. Helicobacter pylori and gastric atrophy— cancer paradoxes. J. Natl Cancer Inst. 1995; 87: 1731–2. 6 Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, Isaacson PG. Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma. Lancet 1991; 338: 1175–6. 7 Megraud F. Antibiotic resistance in Helicobacter pylori infection. Br. Med. Bull. 1998; 54: 207–16. 8 Lee A, Fox JG, Otto G, Murphy J. A small animal model of human Helicobacter pylori active chronic gastritis. Gastroenterology 1990; 99: 1315–23. 9 Marchetti M, Arico B, Burroni D, Figura N, Rappuoli R, Ghiara P. Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 1995; 267: 1655–8. 10 Lee A, O’Rourke J, De Ungria MC, Robertson B, Daskalopoulos G, Dixon MF. A standardized mouse model of Helicobacter pylori infection–Introducing the Sydney strain. Gastroenterology 1997; 112: 1386–97. 11 Ermak TH, Ding R, Ekstein B et al. Gastritis in ureaseimmunized mice after Helicobacter felis challenge may be due to residual bacteria. Gastroenterology 1997; 113: 1118–28. 12 Sutton P, Wilson J, Lee A. Further development of the Helicobacter pylori mouse vaccination model. Vaccine 2000; 18: 2677–85. 13 Marchetti M, Rossi M, Giannelli V et al. Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-liable enterotoxin (LT) as adjuvant. Vaccine 1998; 16: 33–7. 14 Corthesy-Theulaz IE, Hopkins S, Bachmann D et al. Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phoP(c) expressing urease A and B subunits. Infect. Immun. 1998; 66: 581–6. 15 Gomez-Duarte OG, Lucas B, Yan ZX, Panthel K, Haas R, Meyer TF. Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B. Vaccine 1998; 16: 460–71. 16 DiPetrillo MD, Tibbetts T, Kleanthous H, Killeen KP, Hohmann EL. Safety and immunogenicity of phoP/phoQdeleted Salmonella typhi expressing Helicobacter pylori urease in adult volunteers. Vaccine 1999; 18: 449–59. 17 Angelakopoulos H, Hohmann EL. Pilot study of phoP/phoQdeleted Salmonella enterica serovar Typhimurium expressing Helicobacter pylori urease in adult volunteers. Infect. Immun. 2000; 68: 2135–41. 18 Dieterich C, Bouzourene H, Blum AL, Corthesy-Theulaz IE. Urease-based mucosal immunization against Helicobacter heilmannii infection induces corpus atrophy in mice. Infect. Immun. 1999; 67: 6206–9. 19 Goto T, Nishizono A, Fujioka T, Ikewaki J, Mifune K, Nasu M. Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice. Infect. Immun. 1999; 67: 2531–9. 20 Mohammadi M, Czinn S, Redline R, Nedrud J. Helicobacterspecific cell-mediated immune responses display a predominant TH1 phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice. J. Immunol. 1996; 156: 4729–38. 21 Bamford KB, Fan XJ, Crowe SE et al. Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype. Gastroenterology 1998; 114: 482–92. 22 Czinn SJ, Nedrud JG. Oral immunization against Helicobacter pylori. Infect. Immun. 1991; 59: 2359–63. 23 Chen M, Lee A, Hazell SL. Immunisation against Helicobacter infection in a mouse/Helicobacter felis model. Lancet 1992; 339: 1120–1. 24 Czinn SJ, Cai A, Nedrud JG. Protection of germ-free mice from infection by Helicobacter felis after active oral or passive IgA immunization. Vaccine 1993; 11: 637–42. 25 Lee CK, Weltzin R, Thomas WD et al. Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis. J. Infect. Dis. 1995; 172: 161–72. 26 Ferrero RL, Thiberge JM, Kansau I, Wuscher N, Huerre M, Labigne A. The GroES homolog of Helicobacter pylori confers protective immunity against mucosal infection in mice. Proc. Natl Acad. Sci. USA 1995; 92: 6499–503. 27 Wyatt JI, Rathbone BJ, Heatley RV. Local immune response to gastric Campylobacter in non-ulcer disease. J. Clin. Pathol. 1986; 39: 863–70. 28 Weltzin R, Kleanthous H, Guirakhoo F, Monath TP, Lee CK. Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection. Vaccine 1997; 15: 370–6. 29 Ermak TH, Giannasca PJ, Nichols R et al. Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses. J. Exp. Med. 1998; 188: 2277–88. 30 Blanchard TG, Czinn SJ, Redline RW, Sigmund N, Harriman G, Nedrud JG. Antibody-independent protective mucosal immunity to gastric Helicobacter infection in mice. Cell. Immunol. 1999; 191: 74–80. 31 Sutton P, Wilson J, Kosaka T, Wolowczuk I, Lee A. Therapeutic immunization against Helicobacter pylori infection in the absence of antibodies. Immunol. Cell Biol. 2000; 78: 28–30. 32 Pappo J, Torrey D, Castriotta L, Savinainen A, Kabok Z, Ibraghimov A. Helicobacter pylori infection in immunized mice lacking major histocompatibility complex class I and class II functions. Infect. Immun. 1999; 67: 337–41. 33 Mohammadi M, Nedrud J, Redline R, Lycke N, Czinn SJ. Murine CD4 T-cell response to Helicobacter infection—TH1 cells enhance gastritis and TH2 cells reduce bacterial load. Gastroenterology 1997; 113: 1848–57. 34 Saldinger PF, Porta N, Launois P et al. Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection. Gastroenterology 1998; 115: 891–7. 35 Radcliff F, Ramsay AJ, Lee A. Failure of immunisation against Helicobacter infection in IL-4 mice: evidence for the TH2 immune response as the basis for protective immunity. Gastroenterology 1996; 110: A997. 36 Radcliff FJ, Ramsey AJ, Lee A. A mixed Th1/Th2 response may be necessary for effective immunity against Helicobacter. Immunol. Cell Biol. 1997; 75: A90. 37 Akhiani A, Kjerrulf M, Redline R, Nedrud R, Czinn S, Lycke N. Th1 and Th2 cells contribute to protection but are also involved in the development of gastritis. In: Wadstrom T, Anderson L (eds). Third International workshop on pathogenesis and host response in Helicobacter infections, 1–4 July, 1998, Helsingor, Denmark. 1998; Abstract C-1. 38 Jiang B, Jordana M, Xing Z et al. Replication-defective adenovirus infection reduces Helicobacter felis colonization in the mouse in a gamma interferon- and interleukin-12-dependent manner. Infect. Immun. 1999; 67: 4539–44. 39 Akamatsu T, Ota H, Shimizu T et al. Histochemical study of Helicobacter pylori and surface mucous gel layer in various gastric lesions. Acta Histochem. Cytochem. 1995; 28: 181–5. Immunity and H. pylori: A role for mucus? 40 Hazell SL, Lee A, Brady L, Hennessy W. Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. J. Infect. Dis. 1986; 153: 658–63. 41 Shimizu T, Akamatsu T, Sugiyama A, Ota H, Katsuyama T. Helicobacter pylori and the surface mucous gel layer of the human stomach. Helicobacter 1996; 1: 207–18. 42 Schreiber S, Stuben M, Josenhans C, Scheid P, Suerbaum S. In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: Experimental method and results. Infect. Immun. 1999; 67: 5151–6. 43 Younan F, Pearson J, Allen A, Venables C. Changes in the structure of the mucous gel on the mucosal surface of the stomach in association with peptic ulcer disease. Gastroenterology 1982; 82: 827–31. 44 Micots I, Augeron C, Laboisse CL, Muzeau F, Megraud F. Mucin exocytosis: a major target for Helicobacter pylori. J. Clin. Pathol. 1993; 46: 241–5. 45 Takahashi S, Nakamura E, Okabe S. Effects of cytokines, without and with Helicobacter pylori components, on mucus secretion by cultured gastric epithelial cells. Dig. Dis. Sci. 1998; 43: 2301–8. 46 Slomiany BL, Sarosiek J, Bilski J, Slomiany A. Evidence for proteolytic disruption of gastric mucus coat by Campylobacter pylori. S. Afr. Med. J. 1988; 74: 40–1. 47 Slomiany BL, Piotrowski J, Slomiany A. Effect of sucralfate on the degradation of human gastric mucus by Helicobacter pylori proteases and lipases. Am. J. Gastroenterol. 1992; 87: 595–9. 48 Markesich DC, Anand BS, Lew GM, Graham DY. Helicobacter pylori infection does not reduce the viscosity of human gastric mucus gel. Gut 1995; 36: 327–9. 49 Slomiany BL, Piotrowski J, Slomiany A, Konturek JW, Domschke WW. Enhancement in the protective qualities of gastric mucus with combination therapy of ebrotidine and amoxicillin for H. pylori eradication. Gen. Pharmacol. 1998; 31: 227–31. 50 Simon PM, Goode PL, Mobasseri A, Zopf D. Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides. Infect. Immun. 1997; 65: 750–7. 51 Wadstrom T, Hirmo S, Novak H et al. Sulfatides inhibit binding of Helicobacter pylori to the gastric cancer Kato III cell line. Curr. Microbiol. 1997; 34: 267–72. 52 Tzouvelekis LJ, Mentis AF, Makris AM, Spiliasis C, Blackwell C, Weir DM. In vitro binding of Helicobacter pylori to human gastric mucin. Infect. Immun. 1991; 59: 4252–4. 53 Namavar F, Sparrius M, Veerman E, Applemelk BJ, Vandenbrouckegrauls C. Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin. Infect. Immun. 1998; 66: 444–7. 73 54 Hirmo S, Artursson E, Puu G, Wadstrom T, Nilsson B. Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor. J. Microbiol. Methods 1999; 37: 177–82. 55 Genta RM, Gurer IE, Graham DY et al. Adherence of Helicobacter pylori to areas of incomplete intestinal metaplasia in the gastric mucosa. Gastroenterology 1996; 111: 1206–11. 56 Scharfman A, Kroczynski H, Carnoy C et al. Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth. Infect. Immun. 1996; 64: 5417–20. 57 Tomb JF, White O, Kerlavage AR et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997; 388: 539–47. 58 Alm RA, Ling LSL, Moir DT et al. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 1999; 397: 176–80. 59 Ogilvie BM, Love RJ. Co-operation between antibodies and cells in immunity to a nematode parasite. Transplant Rev. 1974; 19: 147–68. 60 Ishikawa N, Horii Y, Nawa Y. Immune-mediated alteration of the terminal sugars of goblet cell mucins in the small intestine of Nippostrongylus brasiliensis-infected rats. Immunology 1993; 78: 303–7. 61 Nawa Y, Miller HRP. Protection against Nippostrongylus brasiliensis by adoptive immunization with immune thoracic duct lymphocytes. Cell. Immunol. 1978; 37: 51–60. 62 Nawa Y, Parish CR, Miller HRP. The protective capacities of fractionated immune thoracic duct lymphocytes against Nippostrongylus brasiliensis. Cell. Immunol. 1978; 37: 41–50. 63 Wells PD. Mucin-secreting cells in rats infected with Nippostrongylus brasiliensis. Exp. Parasitol. 1963; 14: 15–22. 64 Koninkx JFJG, Mirck MH, Hendricks HGCJM, Mouwen JMVM, Van Dijik JE. Nippostrongylus brasiliensis: Histochemical changes in the composition of mucins in goblet cells during infection in rats. Exp. Parasitol. 1988; 65: 84–90. 65 Oinuma T, Abe T, Nawa Y, Kawano J, Suganuma T. Glycoconjugates in rat small intestinal mucosa during infection with the intestinal nematode Nippostrongylus brasiliensis. Adv. Exp. Med. Biol. 1995; 371B: 975–8. 66 Khan WI, Abe T, Ishikawa N, Nawa Y, Yoshimura K. Reduced amount of intestinal mucus by treatment with anti-CD4 antibody interferes with the spontaneous cure of Nippostrongylus brasiliensis-infection in mice. Parasit. Immunol. 1995; 17: 485–91. 67 Nawa Y, Korenaga M. Mast and goblet cell responses in the small intestine of rats concurrently infected with Nippostrongylus brasiliensis and Strongyloides ratti. J. Parasitol. 1983; 69: 1168–70. 68 Lee A, Mellgard B, Larsson H. Effect of gastric acid on Helicobacter pylori ecology. In: Hunt RH, Tytgat GNJ (eds). Helicobacter pylori: Basic mechanisms to clinical cure 1996. Dordrecht: Kluwer Academic Publishers, 1996; 50–63.