Vaccine 22 (2004) 2541–2546

Immunisation againstHelicobacter felis infection protects againstthe development of gastric MALT Lymphoma

Philip Suttona,∗, Jani O’Rourkea, John Wilsona, Michael F. Dixonb, Adrian Leea

a School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australiab Department of Histopathology, University of Leeds, Leeds, UK

Received 8 October 2003; received in revised form 27 November 2003; accepted 19 December 2003

Available online 23 January 2004

Abstract

The formation of mucosa-associated lymphoid tissue (MALT) in response toHelicobacter pylori infection is closely associated withthe development of primary gastric MALT lymphoma.Aim: To examine whether immunisation againstHelicobacter felis can protectagainst development of MALT lymphoma.Results: The majority of control infected mice demonstrated MALT formation (13/15) andfive developed lymphoma. Fifteen immunised mice were protected against bacterial challenge, of which only five had evidence of MALTformation and none developed lymphoma. Interestingly, of the four mice in which immunisation failed, all developed MALT and two ofthese had lymphoma.Conclusion: Effective immunisation againstHelicobacter infection can protect against gastric MALT lymphoma. Toour knowledge this is the first demonstration of vaccination protecting against a bacteria-induced malignancy.© 2004 Elsevier Ltd. All rights reserved.

Keywords: Helicobacter; Vaccine; MALT lymphoma

1. Introduction

Primary gastric lymphomas comprise approximately3–6% of gastric malignancies and are predominantly B lym-phocyte non-Hodgkin’s lymphomas[1]. It was proposedin 1983 that such gastric lymphomas arose within mucosa-associated lymphoid tissue (MALT)[2], which is organ-ised secondary lymphoid tissue containing both T and Bcell zones. However, such structures although commonin the ileum are not a component of normal gastric mu-cosa. Also in 1983 came the first report of a new bacterialpathogen colonising the mucus layer of the gastric mucosa,Helicobacter pylori [3,4]. It was later demonstrated thatlymphoid follicles develop inH. pylori infected persons[5–7] and that the lymphoid tissue formed in response toH.pylori infection is morphologically identical to MALT[8].

It has now been clearly demonstrated that a close link ex-ists between infection withH. pylori and the development ofgastric MALT lymphoma (also termed marginal zone lym-phoma). In 1993, Wotherspoon et al.[9] first reported that

∗ Corresponding author. Present address: Centre for Animal Biotech-nology, University of Melbourne, Melbourne, VIC 3010, Australia.Tel.: +61-3-8344-7152; fax:+61-3-9347-4083.

E-mail address: psutton@unimelb.edu.au (P. Sutton).

eradication ofH. pylori with antimicrobials led to regres-sion of low grade B-cell MALT lymphoma in five out ofsix patients. Now, eradication ofH. pylori is the frontlinetreatment for patients presenting with primary low gradegastric MALT lymphoma. Although approximately 20% ofthe patients with low grade gastric MALT lymphomas donot regress followingH. pylori eradication, early evidencesuggests that these individuals at least do not progress tohigh-grade disease[10].

A key feature of low grade MALT lymphoma is thepresence of destructive lympho-epithelial lesions (LELs).Migration of B-lymphocytes from the marginal zone of afollicle into adjacent epithelium is characteristic of nor-mal MALT. However, when the epithelium is infiltratedand subsequently destroyed by atypical (centrocyte-like)lymphocytes, these lesions are pathognomonic of earlylow grade MALT lymphoma. We previously reported on amouse model ofHelicobacter-induced MALT lymphoma inwhich mice infected withH. felis for 22 months developeddestructive LELs identical to those seen in human MAL-Tomas[11]. Similar to reports in humans, we found thateradication of the bacterial infection in the mouse modelat 20 months post-infection greatly reduced the develop-ment of MALT lymphoma 2–4 months later[12]. Whileinvestigating the long term effects of vaccination against

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2542 P. Sutton et al. / Vaccine 22 (2004) 2541–2546

Helicobacter infection, we noted that mice prophylacticallyimmunised and then challenged withH. felis did not developlymphoid aggregates or follicles in response to infection, incontrast to the infection control group[13]. The reductionin lymphoid follicles occurred even though the immunisedchallenged mice only presented with a reduced level ofbacterial colonisation and not complete eradication of in-fection. We hypothesised that as lymphoid follicles wereessential to the development of gastric MALT lymphoma,prophylactic immunisation may prevent this malignancy.

2. Materials and methods

2.1. Animals

Specific pathogen-free, female BALB/c mice, 7 weeksold, were obtained from the Animal Resource Centre, Perth,Australia. All protocols involving animal experimentationwere approved by the Animal Care and Ethics Committeeat the University of New South Wales.

2.2. Bacterial culture and infection of mice

H. felis, strain CS1 (ATCC 49179), was grown onCampylobacter selective agar (CSA) consisting of 5% (v/v)sterile horse blood in blood agar base no. 2 (Oxoid Ltd.)and Skirrow’s supplement[14]. Plates were incubated inan anaerobic jar with a microaerophilic gas generating kit(code no. BR 56, Oxoid) for 2 days at 37◦C. Helicobacterwere harvested and suspended in brain heart infusion (BHI)and the final concentration was adjusted to approximately109 bacteria/ml. For infection, mice were inoculated intra-gastrically, twice in a 3-day period, with 0.1 ml of bacterialsuspension (approximately 108 bacteria) using a polyethy-lene stomach catheter (0.58 mm internal diameter×0.97 mmouter diameter).

2.3. Immunisation

Helicobacter sonicate was prepared for immunisation byharvesting cells in phosphate buffered saline (PBS) and son-icating with a Branson Sonifier fitted with a microtip (Bran-son Ultrasonics Corporation, Danbury, CT). The proteincontent of the sonicate was determined using the Bio-RadDC protein assay (Bio-Rad, Regents Park, Australia) andthen stored at−70◦C until use. For immunisation, micewere orogastrically dosed with 1 mg ofH. felis sonicate with10 g of cholera toxin (Sigma Chemical Co., St Louis, MO).

2.4. Experimental groups

Group A (control) mice were left untreated as controls forthe spontaneous development of lympho-epithelial lesionsand MALT lymphoma. Group B (infected) mice were sham

treated with PBS before being infected withH. felis. Group C(immunised) mice were immunised withH. felis whole cellsonicate plus cholera toxin (CT) as a mucosal adjuvant. ACT alone control group was not included in this study as wehave found in numerous previous experiments that CT hasno prophylactic efficacy without co-delivery of antigen (datanot shown). Five weeks after the last immunisation, micein groups B and C were challenged withH. felis. The micewere left for 22 months before removal of stomach tissuefor evaluation of bacterial colonisation and histopathology.

2.5. Assessment of histopathology and colonisationby H. felis

At the completion of the experimental protocol all micewere sacrificed. Half of each animal’s stomach was fixed in10% formal buffered saline, embedded in paraffin and sec-tioned in a strip to include the greater curve. Histopathologywas assessed on haematoxylin and eosin stained sections(by MFD) and histological assessment ofH. felis colonisa-tion was performed separately using May-Grünwald-Giemsastained sections (JO). All slides were coded and then graded‘blind’ by light microscopy. The presence of MALT, i.e.lymphoid aggregates and follicles having germinal centres,in the antral and body mucosa was scored on a 0–3 scalerepresenting no, mild, moderate and marked lymphoid infil-tration. A mean score was calculated for each animal.

Likewise the number of atypical LELs was assessed aseither none, a few (1), and moderate numbers (2) while thefinding of multiple, destructive LELs was taken as evidenceof MALT lymphoma (3). The latter group was sub-dividedinto mucosal involvement only (3a) and deeper infiltrationby centrocyte-like cells into the submucosa or beyond (3b).The remaining half of the animal’s stomach was cultured onCSA and incubated as above for 5 days to detect viableH.felis.

3. Results

Prophylactic immunisation withH. felis lysate and CTbefore challenge withH. felis protected the majority of miceagainst bacterial infection. Control mice were confirmed tobe clear ofH. felis infection. Vaccination had an efficacy of79% with 15 of the 19 mice in group C having no detectableH. felis almost 2 years after immunisation as measured byhistological examination of tissue sections and culture ofgastric tissue (Table 1). In contrast, all 15 mice from thenon-immunised, infected group B were infected withH.felis at the end of the experiment as determined by bothmicroscopy and culture. This latter group demonstrates theefficiency of our infection procedure, as 100% of the groupB mice were successfully infected.

Gastric sections from all mice were processed forhistopathological examination (examples shown inFig. 1).Only one mouse in group A showed any increase in lympho-

P. Sutton et al. / Vaccine 22 (2004) 2541–2546 2543

Table 1Effect of prophylactic immunisation againstHelicobacter felis on devel-opment of MALT lymphoma

cytic infiltration in the mucosa and this was mild (Table 1).No LELs or low grade MALT lymphomas were observedin the control mice. In contrast, 13/15 of the infected micehad evidence of MALT formation, while 11/15 had eitheratypical LELs (6) or low grade MALT lymphoma (5).

Mice protected againstH. felis infection by prophylacticimmunisation had a significantly reduced level of MALT de-velopment (P < 0.01; Fisher’s exact test) compared to thenon-immunised infected mice and none showed evidenceof lymphoma. Three of the nineteen immunised mice hadatypical LELs. Importantly, the three mice in the immunisedgroup which had LELs were those in which immunisationhad failed and were therefore infected withH. felis. Simi-larly, although 9/19 of the immunised mice had some de-gree of MALT formation, four of these mice were infectedwith H. felis. Thus, if the 15 immunised mice which weresuccessfully protected againstH. felis infection by vaccina-tion are considered separately, 5/15 had evidence of MALTformation but none had LELs or lymphoma.

There was no apparent correlation between bacterial den-sity and MALT formation in infected mice. All infectedmice had high infection scores under histological examina-tion (data not shown), although this method for assessingH. felis colonisation of mouse gastric tissue, though the bestavailable, is semi-quantitative and does not allow meaning-ful statistical analysis.

For more precise quantification, it is appropriate to con-sider the four failed immunised mice as part of the infectedgroup B. If this is done, then 74% (14/19) of the infectedmice developed atypical LELs or frank lymphoma, com-pared with none (0/15) in the protectively immunised mice,demonstrating that immunisation produced highly signifi-cant protection againstHelicobacter-induced formation oflymphoma and its precursors (P < 0.0001; Fisher’s exacttest).

4. Discussion

The normal healthy stomach is devoid of lymphoid tis-sue, apart from a few small basal aggregates in the corpus.The situation changes, however, when the human gastricmucosa is infected withH. pylori, as is the case in approx-imately half the world’s population. This chronic bacterialinfection which usually commences during childhood and ismaintained for decades, produces an inflammatory responsethat is marked by the infiltration into the gastric mucosa oflarge numbers of lymphocytes and variable numbers of neu-trophils. It is this active, chronic inflammatory response thatis responsible for the majority of cases of peptic ulcers andis a predisposing factor in most gastric adenocarcinomas.

The stimulation of an immune response in the gastricmucosa induces first the formation of aggregates of lympho-cytes which become organised over time into follicular lym-phoid tissue with germinal centres. The acquired lymphoidtissue is identical to MALT seen at other sites. The forma-

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Fig. 1. Examples of the histopathology seen in the animals 22 months after prophylactic immunisation and bacterial challenge. Uninfected age-matchedcontrol animals (A) and mice in which prophylactic immunisation was successful in preventing bacterial colonisation (B) showed normal gastric mucosawith no significant influx of inflammatory cells (magnification 1000×). By comparison, mice in the controlH. felis infection group (C) and those animalsin which immunisation was unsuccessful in preventing colonisation (D) showed an expansive increase in inflammatory cells, primarily lymphocytes,resulting in the formation of MALT in the mucosa often extending to the submucosa (magnification 2000×). In several instances atypical lymphocyteswere found invading and destroying the gastric epithelium resulting in the formation of destructive lympho-epithelial lesions, a feature of MALT lymphoma(indicated by∗ in the insets of C and D; magnification 3500×).

tion of gastric MALT triggered by chronic inflammationis an essential first step in the development of gastric lym-phoma[15]. The organised lymphoid tissue creates an en-vironment where proliferating CD4+ T-cells, stimulated by

Helicobacter antigens, provides T-cell help which supportsthe ongoing inflammatory cell response[16,17]. In someindividuals, thisHelicobacter-specific T-cell help also pro-motes the development and proliferation of B-lymphocytes

P. Sutton et al. / Vaccine 22 (2004) 2541–2546 2545

with specificity for autologous antigens[16,18]. Prolongedlymphocytic proliferation due to antigen stimulation canlead to the emergence of a neoplastic clone, arising from thememory B-cells of the marginal zone of the follicle. The newaggressive clone gradually replaces the normal lymphoidcells in the lamina propria of the mucosa, colonises germi-nal centres, invades epithelium and infiltrates through themuscularis mucosae features indicating an unequivocal pro-gression to lymphoma[15,19]. If performed early enough,eradication ofHelicobacter infection, which removes anti-genic stimulation of T-cells and, in turn, helper-cell stimula-tion of the lymphoma B-cells, can lead to resolution of manylow grade lymphomas[9]. However, there does appear to bea point of no return when T-cell help is no longer requiredto support continued proliferation of the lymphoma cells,at which time eradication ofHelicobacter infection has noeffect [19,20].

While investigating the effect of prophylactic immuni-sation on post-immunisation gastritis in a mouse modelof Helicobacter infection we made the observation thatimmunised mice, in contrast to infected mice, did not de-velop lymphoid aggregates following challenge withH.felis, even though the immunised challenged mice werestill infected with bacteria, albeit to a lower degree[13].This suggested that prophylactic immunisation, althoughnot completely protecting againstHelicobacter infection,modified the immune response in such a way that inhibitedthe bacterial drive that induced aggregation of lymphocytesand hence formation of gastric MALT. We hypothesisedthat immunisation would thus protect against developmentof Helicobacter-induced MALT lymphomas. As we hadpreviously developed a mouse model of gastric MALTlymphoma[11,12] we set out to test this hypothesis.

In this study, we demonstrated that prophylactic immu-nisation protected the majority of mice againstH. felis asmeasured 22 months after bacterial challenge, and foundonce again that this protection significantly reduced theformation of gastric lymphoid tissue. Additionally and ashypothesised, immunisation prevented the formation ofatypical lympho-epithelial lesions, a precursor of MALTlymphoma. Although group sizes were probably too low toperform meaningful statistics, it is also interesting that noneof the protectively immunised mice developed low gradelymphoma in contrast to the infected mice. It could beargued that the 33% of successfully immunised mice withMALT remain at risk of lymphoma development and thatthe experiment was of insufficient duration to demonstrateprotection in this group. However, it must be borne in mindthat the end-point of 22 months is approaching the naturallife-span of these mice.

Four of the nineteen immunised/challenged mice werefound to be infected withH. felis. As it transpired, thefailure of immunisation in these mice proved fortuitousas they exemplified the effect of protective immunisationagainstHelicobacter infection on development of LEL andMALT lymphoma. None of the 15 immunised mice which

were negative forHelicobacter infection developed atypicalLELs or lymphoma. In contrast, two of the four immunisedmice in which bacterial infection was present at 22 monthshad early lymphoma.

In conclusion, by comparing the gastric histopathologyof protectively immunised mice with that of infected mice,we have demonstrated that prophylactic immunisationagainstH. felis infection can protect against the develop-ment of gastric MALT lymphoma. To our knowledge thisis the first demonstration of vaccination protecting againsta bacteria-induced malignancy.

Acknowledgements

This work was funded by the National Health and Med-ical Research Council of Australia. We would like to giveour appreciation to Angelina Enno for her histological as-sistance.

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