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  • 1. GASTROENTEROLOGY 1997;113:1118–1128 Gastritis in Urease-Immunized Mice After Helicobacter felis Challenge May Be Due to Residual Bacteria THOMAS H. ERMAK,* RU DING,* BRUCE EKSTEIN,* JOSEPH HILL,‡ GWENDOLYN A. MYERS,* CYNTHIA K. LEE,* JACQUES PAPPO,* HAROLD K. KLEANTHOUS,* and THOMAS P. MONATH* *OraVax, Inc., Cambridge, Massachusetts; and ‡Clemson Livestock Diagnostic Lab, Clemson University, Columbia, South Carolina Background & Aims: Oral immunization with recombi- in the gastric mucosa. Protection is associated with mild nant Helicobacter pylori urease (rUre) coadministered inflammation in the antrum characterized by the presence with a mucosal adjuvant protects mice against chal- of immunoglobulin A–secreting B cells and CD4//ab- lenge with Helicobacter felis. In this study, the duration TCR/ T cells.10 Several months after protection has been of protection and gastritis after challenge were charac- achieved, immunized mice may exhibit a lymphocytic terized at sequential time intervals up to 1 year. Meth- infiltration into the corpus,7 raising questions as to ods: Outbred Swiss–Webster mice were orally immu- whether immunization was effective in resolving Helico- nized with rUre plus adjuvant and examined for the presence of H. felis infection and leukocyte infiltration bacter-induced gastritis and if immunopathologic effects into the gastric mucosa. Results: When defined by gas- were induced by the vaccine. tric urease activity, 70%–95% of rUre-immunized mice H. pylori–induced gastritis in humans is characterized were protected for between 2 and 57 weeks. Challenge by infiltration of lymphocytes, plasma cells, and poly- with H. felis increased the inflammatory response in morphonuclear leukocytes11 – 13 and, in severe cases, is the gastric mucosa of rUre-immunized mice, which also associated with epithelial changes consisting of parietal had elevated CD4/ and CD8/ T cells. The CD8/ cells cell loss, hyperplasia of the surface epithelium (foveolar represented a population of gastric intraepithelial cells, hyperplasia), and intestinal metaplasia.14 Murine H. felis– which expressed the mucosal aE-integrin. Epithelial induced gastritis is characterized by a similar but primar- changes consisting of parietal cell loss and hyperplasia ily lymphocytic infiltrate as well as formation of microab- of the epithelium occurred in approximately 20% of the scesses and cystic glands.4 mice. Antimicrobial triple therapy significantly de- creased the degree of gastritis and epithelial alteration In this study, the effect of rUre immunization on the in the stomach. Conclusions: These results indicate duration of protection was examined for periods of up to that oral immunization of mice with rUre produces a a year after challenge with H. felis, with special attention long-lasting inhibition of H. felis infection but that resid- to the kinetics of antral and corpus gastritis, the pheno- ual bacteria may produce a persistent lymphocytic in- type of infiltrating T cells, and the epithelial cell compo- filtration under these experimental conditions. sition of the gastric mucosa. We found that 70%–95% of mice were protected up to 57 weeks after challenge as defined by gastric urease activity. In addition, Ç50% H elicobacter pylori is a gram-negative spiral bacterium that colonizes the gastric mucosa of humans, caus- ing chronic superficial gastritis and peptic ulcers; it has of rUre-immunized mice remained free of infection and had no gastritis. Approximately 10% of rUre-immunized been implicated in adenocarcinoma and atrophy of the mice developed a chronic lymphocytic gastritis, charac- mucosa.1,2 Helicobacter felis colonizes the gastric mucosa terized by both CD4/ and CD8/ T cells. However, clear- of mice and has been used as a convenient model for ance of H. felis by triple antimicrobial treatment after studying immunity in the development of oral vaccines challenge prevented gastritis in all mice, implicating re- against H. pylori.3 – 6 Oral immunization with recombi- sidual bacteria as the cause of lymphocytic infiltration nant H. pylori urease (rUre) coadministered with a muco- after rUre immunization. sal adjuvant such as cholera toxin (CT) or Escherichia coli heat-labile enterotoxin (LT) induces a mucosal immune Abbreviations used in this paper: CT, cholera toxin; DAB, diamino- response, protects mice against challenge with H. felis, benzidine; HBSS, Hanks’ balanced salt solution; IEL, intraepithelial and clears bacteria from gastric tissue of previously in- lymphocyte; LT, Escherichia coli heat-labile enterotoxin; rUre, recom- binant Helicobacter pylori urease. fected mice.7 – 10 Prophylactically immunized mice chal- 1997 by the American Gastroenterological Association lenged by H. felis are protected from bacterial infection 0016-5085/97/$3.00 / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 2. October 1997 ORAL IMMUNIZATION WITH RECOMBINANT UREASE 1119 Materials and Methods pathologic, and immunohistochemical analyses. A longitudinal segment including the antrum and corpus plus a piece of Animals, Immunization, and Challenge attached intestine was fixed in 10% neutral buffered formalin, Specific pathogen–free, 8-week-old outbred female later routinely processed, embedded in paraffin, sectioned at 5 Swiss–Webster mice that were free of Helicobacter muridarum mm, and stained with H&E. For immunohistochemical analy- were obtained from Taconic Farms, Inc. (Germantown, NY). sis, gastric segments were mounted in O.C.T. embedding com- All procedures were conducted with approval of the OraVax pound (Miles Scientific, Naperville, IL) and quick-frozen in Institutional Animal Care and Use Committee. Freon 22 cooled to its freezing point by liquid nitrogen. Mice were given four oral immunizations at weekly intervals of 25–200 mg rUre (OraVax Development Section, OraVax, Urease Activity Inc., Cambridge, MA) with either 10 mg CT or 5 mg LT The presence of H. felis in gastric tissue was assessed adjuvant in 0.2 mL phosphate-buffered saline (PBS) or adju- by urease activity measured spectrophotometrically using a vant alone in PBS. rUre- and CT- or rUre- and LT-immunized colorimetric urease test. Antral segments (one quarter of the mice not challenged with H. felis served as controls. Mice entire antrum) from each mouse were dissected free of corpus were challenged 2–4 weeks after the last immunization with and intestine, placed in 0.5 mL of urea broth containing phenol a single intragastric dose of 1 1 107 H. felis organisms (CS- red, and incubated as a whole piece of tissue for 4 hours at 1 strain). This dose represents 103 times the 90%-infectious room temperature. Thereafter, samples were centrifuged, and intragastric dose.9 200 mL of the supernatant was used to determine the ab- sorbance at 550 nm. The limit of sensitivity of this assay was Protocols to Study Protection and previously determined to be Ç5 1 103 H. felis organisms. Gastritis Using CT or LT as Adjuvants A 7-month study was performed to examine protection Histopathology and gastritis in groups of 10 rUre- and 10 CT-immunized Bacterial colonization, gastritis, and epithelial changes mice 2, 10, and 30 weeks after challenge. A larger, year-long were evaluated on randomized, coded histological sections of study was also conducted using the more clinically relevant gastric mucosa by an experienced veterinary pathologist (J.H.) LT as a mucosal adjuvant. Groups of 25 rUre-immunized and who was blinded to the experimental design and number of 20 LT-immunized mice were challenged 4 weeks after immu- animal groups. H. felis organisms were visualized in gastric nization and examined 2, 10, 17, 34, and 57 weeks later. sections with a modified Steiner silver stain (Sigma).15 The number of H. felis organisms per longitudinal section from the Protocol to Study Gastric Intraepithelial intestine through the entire antrum and corpus was scored as Lymphocytes follows: 0, no bacteria; 1, 1–20 bacteria; 2, 21–50 bacteria; Intraepithelial lymphocytes (IELs) were isolated from 3, 51–100 bacteria; or 4, ú100 bacteria. gastric tissue of mice immunized with rUre and either CT or For evaluation of gastritis, H&E-stained sections were LT as an adjuvant and killed 4–6 months after challenge. scored based on the intensity of the infiltration of lymphocytes, plasma cells, and neutrophils.9 Grades were defined as follows: Protocol to Examine the Role of Residual 0, none; 1, a few leukocytes scattered in the deep mucosa; 2, Bacteria in Gastritis moderate numbers of leukocytes in the deep to mid mucosa and occasional neutrophils in gastric glands (microabscesses); Groups of 10 mice were immunized with 25 mg rUre 3, dense infiltrates in the mid to deep mucosa, a few microab- and 10 mg CT. Starting 2 weeks after challenge and continuing scesses, and one or two lymphoid aggregates; and 4, dense, through the 4th week after challenge, mice received 0.675 diffuse infiltrates throughout the lamina propria and into the mg metronidazole, 1.5 mg tetracycline, and 0.185 mg Pepto- submucosa, with prominent lymphoid aggregates, and several Bismol (Procter and Gamble, Cincinnati, OH) daily for 14 microabscesses. days or no antimicrobial treatment at all. Control groups for The degree of epithelial change was scored as follows: 0, the experiment received (1) no immunization, challenge, and none; 1, small, focal areas of parietal cell loss in the corpus and/ triple therapy, (2) no immunization, challenge, and no triple or hyperplasia of the surface epithelium; 2, epithelial changes therapy, or (3) CT immunization, challenge, and no triple throughout 75% of the mucosa; 3, epithelial changes through- therapy. Gastric tissue was sampled 10 and 20 weeks after out the mucosa plus one to three microabscesses or cystic challenge and scored for gastritis and epithelial changes, and glands; or 4, epithelial changes throughout the mucosa plus half the samples from rUre-immunized mice were processed four or more microabscesses or cystic glands. for immunohistochemical evaluation of T-cell subsets. Metaplasia of surface mucus cells was determined in histo- logical sections stained with alcian blue. Control sections were Tissue Analysis stained with periodic acid–Schiff reagent. The degree of meta- The stomach was dissected along the lesser curvature, plasia was scored as follows: 0, none; 1, a few isolated cells or divided into quarters, and subjected to urease activity, histo- one focal area of alcian blue–positive mucous cells; 2, at least / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 3. 1120 ERMAK ET AL. GASTROENTEROLOGY Vol. 113, No. 4 two focal areas of metaplasia in the surface epithelium or cystic Results glands; or 3, metaplasia throughout ú50% of the epithelium. Duration of Protection, Histopathology, and T Cells Using CT as an Adjuvant Analysis of T-Cell Phenotype Cryosections were blocked with avidin-biotin– Oral immunization with rUre and CT resulted in blocking reagents (Vector Laboratories, Burlingame, CA) and 60% protection against H. felis infection through 30 sequentially incubated with rat monoclonal antibodies against weeks after challenge, but 6 of 15 rUre-immunized mice mouse CD4 (clone RM4-5), CD8 (clone 53-6.7), aE-integrin had gastritis scores of ¢3 (only 1 of 15 CT-immunized (M290), b7 -integrin (clone M293), Vb2 (clone B20.6), Vb6 mice had severe gastritis). Analysis of the CD4/ and (clone RR4-7), Vb7 (clone TR310), or Vb14 (clone 14-2) (Phar- CD8/ cells from mice at 30 weeks after challenge showed mingen, San Diego, CA). Sections were then incubated with that rUre-immunized mice with gastritis scores of ¢3 biotinylated rabbit anti-rat immunoglobulin G (Vector Labo- had CD4/ and CD8/ cells in the gastric mucosa, but ratories), followed by horseradish peroxidase conjugated to an that CT-immunized mice had only CD4/ cells (data not avidin-biotin complex (Vector Laboratories), diaminobenzidine shown). Because of the small number of animals exam- (DAB), and methyl green. Control sections were incubated ined, a larger, quantitative experiment was performed without primary monoclonal antibody. The number of CD4/ using LT instead of CT as an adjuvant because LT had or CD8/ cells was counted by one of the authors (T.H.E.) in greater potential for eventual use in human vaccines. 0.5-mm segment lengths of gastric mucosa and expressed per square millimeter of field. Duration of Protection, Histopathology, and T Cells Using LT as an Adjuvant Two-Color Flow Cytometry of Gastric IELs Duration of protection. The year-long study in Gastric and intestinal tissues were incubated in Ca2/- rUre- plus LT-immunized mice was composed of four and Mg2/-free Hanks’ balanced salt solution (HBSS) con- experimental groups: (1) rUre- plus LT-immunized and taining ethylenediaminetetraacetic acid (EDTA), and the H. felis–challenged; (2) LT-immunized and H. felis– dispersed IELs were purified by Percoll gradient centrifuga- tion.16,17 Gastric and intestinal IEL were incubated simultane- challenged; (3) rUre- plus LT-immunized but not chal- ously with fluorescein isothiocyanate–conjugated and biotin- lenged; and (4) LT-immunized but not challenged. As conjugated antibodies in V-bottomed microtiter plate wells determined by the absence of gastric urease activity, (106 cells/well) and then with the appropriate dilution of strep- 70%–95% of mice immunized with rUre remained pro- tavidin-conjugated phycoerythrin (Becton Dickinson, Moun- tected at various time intervals up to 57 weeks after tain View, CA). The stained lymphocytes were fixed in 1% challenge (Figure 1A, rUre / LT group). In contrast, paraformaldehyde and analyzed (10,000 cells) using an Epics ú95% of LT-immunized mice remained infected at the XL (Coulter Corp., Miami, FL), from which the percentage of maximal detectable level over this time interval (Figure each lymphocyte subpopulation was determined. 1A, LT group). The kinetics of protection as defined by histological examination for H. felis in gastric tissue Detection of Gastric Autoantibodies sections was similar (Figure 1B). However, some mice Acetone-fixed cryosections of gastric mucosa from nor- with negative gastric urease activity had low bacterial mal mice were incubated with sera (1:100 dilution) from rUre- counts (õ20 bacteria per section compared with ú100 plus CT-immunized mice (n Å 8) killed 7 months after H. per section for infected controls) and, therefore, slightly felis challenge. Sections were subsequently incubated with lower levels of full protection (30%–82%). horseradish peroxidase–conjugated sheep anti-mouse immu- Histopathology. Unchallenged mice had only a noglubulins (Amersham Corp., Arlington Heights, IL), fol- few infiltrating leukocytes in the gastric mucosa (Figure lowed by DAB and methyl green. Control sections were labeled 2). However, all mice challenged with H. felis had a without the test sera. Unchallenged rUre- plus CT-immunized greater density of cells in the antrum, and rUre-immu- mice (n Å 4) and CT-immunized H. felis–challenged mice (n nized challenged mice had a greater infiltration in the Å 4) at the same time interval served as experimental controls. corpus. Lymphocytes were the predominant cell type, infiltrated the lamina propria from the muscularis mu- Statistics cosa to the surface epithelium, and sometimes formed Statistical analyses were performed with JMP software nodulelike aggregates in the deep mucosa and submu- using Fisher’s Exact Test for dichotomous variables, the Wil- cosa. IELs were also found in the mucosa of rUre-immu- coxon’s/Kruskal–Wallis (rank sums) test for continuous or nized mice. Although infiltration of lymphocytes ordinal variables, or linear regression. increased in response to challenge, Ç50% of rUre-immu- / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 4. October 1997 ORAL IMMUNIZATION WITH RECOMBINANT UREASE 1121 nized mice cleared the infection and had no corpus gastri- tis (scores of 0 or 1) during the year thereafter (Figure 2B). There was no significant increase in the overall degree of inflammation as a function of time in either group, but gastritis scores of ¢3 occurred at every time interval between 10 and 57 weeks in rUre-immunized mice and at 57 weeks in LT-immunized mice (Figure 2). Ten percent of rUre-immunized mice but only 3% of mice in the LT group had corpus gastritis scores of ¢3 (P Å 0.033). Mice with scores of ¢3 had extensive Figure 2. Leukocytic infiltration in the (A ) antrum and (B ) corpus of rUre-immunized and LT-treated mice at sequential time intervals after H. felis challenge. (A ) Challenged mice had greater numbers of leuko- cytes at each time interval (rUre-immunized mice, P õ 0.0075, chal- lenged vs. unchallenged groups at each time interval; LT group, P õ 0.0360, challenged vs. unchallenged groups at each time interval; Wilcoxon’s rank sum test). (B ) rUre-immunized mice had higher scores than LT-immunized controls after H. felis challenge at each time inter- val except 57 weeks (significance at 2–34 weeks, P õ 0.025). Among rUre-immunized mice, challenged groups had higher scores than un- challenged groups (significance at 2–34 weeks, P õ 0.0025; Wil- coxon’s rank sum test). The ’’No H. felis‘‘ group includes all unchal- lenged mice from 2 to 57 weeks after challenge (each data point Å 2 mice). Figure 1. (A ) Urease activity and (B ) H. felis bacterial scores in gastric tissue at sequential time intervals after challenge. Mice were immu- infiltration of lymphocytes in the mucosa and well-devel- nized with 100 mg rUre plus 5 mg LT. (A ) By urease assay, levels of oped submucosal lymphoid aggregates in the corpus protection in rUre-immunized mice were as follows: 2 weeks, 96% (24 of 25); 10 weeks, 70% (14 of 20); 17 weeks, 68% (15 of 22); 34 (Figure 3). The gastritis in rUre-immunized mice did weeks, 95% (18 of 19); and 57 weeks, 88% (15 of 17); all time not diminish during the subsequent year; however, by intervals, P õ 0.0001, two-tailed Fisher’s Exact Test, rUre-immunized 57 weeks after challenge, both rUre-immunized and LT- vs. LT-immunized mice. The urease assay only assessed H. felis colo- nization in the antrum. (B ) By Steiner stain histology, levels of protec- treated mice had roughly equivalent gastritis scores. tion in rUre-immunized mice were as follows: 2 weeks, 60% (15 of Corpus epithelium. Some mice challenged with 25); 10 weeks, 30% (6 of 20); 17 weeks, 62% (13 of 21); 34 weeks, H. felis also showed epithelial changes in the corpus. 79% (15 of 19); and 57 weeks, 82% (14 of 17); all time intervals, P õ 0.01, two-tailed Fisher’s Exact Test, rUre-immunized vs. LT-immunized Parietal cell loss and hyperplasia of the surface epithelium mice. By histology, the entire antrum and corpus was evaluated. Mice were the most common alterations, but microabscesses were considered protected by the urease assay if their optical density and cystic glands were also observed (Figure 4). Approxi- value at 550 nm was within 2 SD of the mean value of unchallenged mately 20% of rUre-immunized mice but only 3% of mice and by a histology score of 0. The ’’No H. felis‘‘ group includes all unchallenged mice from 2 to 57 weeks after challenge (each data LT-immunized mice had an epithelial score of ¢3 (P Å point Å 2 mice). 0.001; Table 1). A modest correlation was seen between / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 5. 1122 ERMAK ET AL. GASTROENTEROLOGY Vol. 113, No. 4 gastritis and epithelial scores in the corpus (r2 Å 0.528), areas of metaplasia only were found in the corpora of suggesting that perhaps the degree of leukocytic infiltra- mice that had been challenged with H. felis (Table 1). tion was a factor but not the only determinant of epithe- Metaplasia was found in surface epithelium (Figure 4C) lial change. and in epithelium lining cystic glands. Neither metapla- Occasional metaplasia of surface mucous cells was sia in the antrum nor formation of intestinal-type goblet found in the corpora of all groups, but focal or extensive cells was evident in any of the mice. CD4/ and CD8/ T cells. Only small numbers of scattered CD4/ T cells were found in the mucosa of mice not challenged with H. felis (Figure 5). After challenge, however, larger numbers of CD4/ cells were apparent in Figure 3. Lymphocytic infiltration in the corpus of H. felis –challenged mice. H&E-stained histological sections. (A ) Unimmunized (LT) Figure 4. Epithelial alteration in the corpus of an H. felis –challenged, mouse: gastritis score, 1; epithelial score, 0. Corpus is composed of rUre-immunized mouse (gastritis score, 3; epithelial score, 3). (A ) surface epithelium (S ), gastric pits lined by mucous and parietal cells H&E-stained histological section. Parietal and chief cells are de- (P ), and chief cells (C ) in the basal glands (original magnification 901). creased in cystic glands containing mucus and cell debris and lined M, muscularis mucosa. (B and C ) Gastritis, epithelial hyperplasia, and by hyperplastic epithelium (arrows ) (original magnification 2101). (B ) dilated glands in rUre-immunized mouse: gastritis score, 3; epithelial Alcian blue–stained histological section. Mucous cell metaplasia score, 3. The corpus shows extensive infiltration of lymphocytes in (arrows ) in the corpus of rUre-immunized mice (metaplasia score, 2). lamina propria (asterisks ), submucosal lymphoid aggregates (L ), and The surface epithelium contains alcian blue–reactive mucus (original parietal cell loss (original magnification 901). magnification 2101). / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 6. October 1997 ORAL IMMUNIZATION WITH RECOMBINANT UREASE 1123 Table 1. Alteration of Gastric Epithelium in the Corpus of found in protected rUre-immunized mice that did not rUre- Plus LT-Immunized and LT-Immunized Mice at develop a lymphocytic gastritis and in unprotected LT- Sequential Time Intervals After H. felis Challenge immunized mice (Figures 2 and 5). Weeks after challenge Expression of mucosal integrins. To ascertain whether gastric T cells belong to a mucosal or a systemic Group 2 10 17 34 57 lineage, gastric tissue was screened for the presence of a Epithelial score ¢3 mucosal integrins. The aE- and b7 -integrins are present rUre / LT/H. felis challenge 6/25 3/20 3/22 3/19 6/17 LT/H. felis challenge 0/24 0/23 0/24 1/24 3/21 as part of the aEb7 heterodimer on intestinal IELs18,19 rUre / LT 0/10 0/9 0/8 0/9 0/7 and mediate adhesion with E-cadherin on epithelial LT 0/10 0/10 0/7 0/8 0/7 cells.20,21 The b7 -integrin is also present as part of the Metaplasia score ¢2b rUre / LT/H. felis challenge 2/25 3/20 5/22 5/19 6/17 a4b7 Peyer’s patch homing receptor.22 In rUre-immu- LT/H. felis challenge 0/24 0/23 0/24 1/24 3/21 nized mice, b7 -integrin–positive cells were localized a throughout the mucosa and were more numerous than Epithelial score ¢3 defined as hyperplasia of surface epithelium or parietal cell loss throughout the mucosa plus 1–3 microabscesses either CD4/ or CD8/ cells, whereas aE-integrin–posi- or cystic glands. tive cells had a similar distribution to, but a slightly higher density than, CD8/ cells (Table 2). Gastric aE- b Metaplasia ¢2 defined as alcian blue–positive cells in 2 or more focal areas of surface epithelium or cystic glands. Unchallenged rUre / LT and LT mice had no metaplasia scores ¢2. integrin–positive cells were also found in LT-immunized mice challenged with H. felis, indicating that a popula- tion of gastric CD80 cells (20%–40%) also expressed aE-integrin (Table 2). the gastric mucosa of rUre-immunized mice compared To examine whether CD8/ cells in gastric tissue repre- with the other experimental groups (Figure 5A and B). sented a population of aE-integrin–positive IELs, iso- CD4/ cells localized in the lamina propria between gas- lated gastric and intestinal CD8/ IELs were examined tric pits, in the subglandular regions of the deep mucosa by flow cytometry (Figure 8). Two-color plots of intesti- above the muscularis mucosa, and in submucosal nal and gastric IELs were very similar, and Ç90% of lymphoid aggregates below the muscularis mucosa (Fig- gastric CD8/ IELs expressed aE-integrin. ure 6A and B). In the corpus, the highest number of TCR b-chain usage. An analysis of TCR b-chain CD4/ cells occurred in rUre-immunized mice 17–57 expression by T cells in gastric tissue (Vb2 , Vb6 , Vb7 , and weeks after challenge, with a maximum density more Vb14 ) revealed a significant infiltration of Vb6/ cells in the than four times greater than that observed in the LT- gastric mucosa of rUre-immunized but not LT-immunized immunized group (Figure 5). mice. Vb6/ cells had a similar distribution as CD8/ cells, CD8/ cells were rare in the mucosa of all mice that were on the average only half as numerous, and were were not challenged and in LT-immunized mice chal- absent if CD8/ cells were not present (Table 2). lenged with H. felis (Figure 5C and D). In rUre-immu- nized mice, however, CD8/ cells were noted in the an- Role of Residual Bacteria in Production of trum of Ç75% and in the corpus of ú85% after Gastritis challenge with H. felis. The highest numbers of CD8/ Tests were performed to examine whether autoim- cells were found between 10 and 57 weeks after chal- munity or impurities in the urease might be responsible lenge, and the density of CD8/ cells and CD4/ cells for the leukocytic infiltration. Analysis of sera from rUre- correlated in the corpus (r2 Å 0.681) but not the antrum immunized mice at 7 months after challenge (8 mice (r2 Å 0.116). CD8/ cells infiltrated the lamina propria, receiving CT as an adjuvant) did not reveal anti–parietal were prominent within the epithelium as IELs (Figure 6C cell antibodies reactive with normal mouse gastric tissue. and D), and were also present in submucosal lymphoid Likewise, immunization of mice with E. coli GroEL (a aggregates. potential rUre contaminant) and CT was not protective Protected vs. unprotected rUre-immunized mice. and produced no greater gastritis than CT treatment over An analysis of unprotected vs. protected rUre-immunized a 10-week observation period after challenge (data not mice pooled from time intervals from 10 to 57 weeks shown). revealed that no unprotected mice had elevated gastritis Because significant lymphocytic infiltration was ob- scores (no gastritis scores ¢3 or T cells in either the served only in mice with no or only a few H. felis organ- antrum or corpus) (Figure 7). Thus, T-cell infiltration in isms (õ20 per section), we examined whether residual rUre-immunized unprotected mice was similar to that bacteria below the level of detection of the urease assay / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 7. 1124 ERMAK ET AL. GASTROENTEROLOGY Vol. 113, No. 4 Figure 5. Kinetics of CD4/ cell and CD8/ cell infiltration in the (A and C ) antrum and (B and D ) corpus of H. felis –challenged mice. (A and B ) rUre-immunized mice (protected only) had greater numbers of CD4/ T cells than LT-control mice (P õ 0.02; antrum CD4/ cells at 2, 10, and 34 weeks and corpus at 34 weeks, rUre / LT vs. LT). (C and D ) rUre-immunized mice had elevated CD8/ cell counts in gastric mucosa at each time interval from 10 through 57 weeks (P õ 0.01; rUre / LT vs. LT). CD8/ cells were rare to absent in unchallenged rUre-immunized mice and LT-treated mice challenged with H. felis. P values given in the figure are for all mice from 10 to 57 weeks after H. felis challenge (rUre / LT vs. LT). or histological study might be responsible for gastritis. that were given either CT or no immunization, and had rUre-immunized mice that received CT as the adjuvant a higher score (P Å 0.0387) (only in the corpus) than were used in this study based on the observation that mice that received no immunization or adjuvant before CT produced slightly greater lymphocytic infiltration antimicrobials (data not shown). Likewise, the density than LT. For antimicrobial treatment, mice were given of CD4/ and CD8/ cells infiltrating the antrum was 14 twice-daily treatments of metronidazole, tetracycline, significantly reduced (P õ 0.01) by antimicrobial treat- and Pepto-Bismol between 2 and 4 weeks after H. felis ment, and the induction of CD4/ and CD8/ cells in the challenge. This time interval was chosen to allow lym- corpus was prevented (0/9 in antimicrobial-treated mice phocytes time to infiltrate the antrum and inhibit bac- vs. 3/11 in rUre-immunized mice that received no ther- terial colonization before antimicrobial treatment. By apy; significance not achieved) (Table 3). 10–20 weeks after challenge, mice that underwent anti- microbial therapy had significantly lower gastritis (P õ Discussion 0.01) and epithelial scores (P õ 0.05) than mice under- The present study indicates that immunization of going no antimicrobial treatment (Table 3), had gastritis mice with rUre produces a long-lasting reduction in H. or epithelial scores equivalent to those of challenged mice felis colonization of gastric mucosa up to 1 year after / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 8. October 1997 ORAL IMMUNIZATION WITH RECOMBINANT UREASE 1125 Figure 6. Distribution of T cells in the gastric mucosa of rUre-immunized mouse with gastritis and epithelial scores of 3. Cryosection of the corpus 57 weeks after challenge with H. felis. (A ) CD4/ T cells localize in the lamina propria below surface epithelium, between gastric pits, adjacent to the muscularis mucosa (M ), and in deep mucosal lymphoid aggregates (L ). CD4/ cell counts in this mouse were 140 cells/mm2 field. (B ) Higher-power view showing CD4/ T cells in the lamina propria (asterisk ). (C ) CD8/ T cells localize in the gastric epithelium and in the lamina propria between gastric pits. CD8/ cell counts in this mouse were 160 cells/mm2 field. (D ) Higher-power view showing CD8/ IELs (arrows ) in the surface epithelium. (Original magnifications: A and C, 901; B, 1801; D, 3601.) challenge. H. pylori urease, a major protein required for proportion of LT-immunized mice infected with H. felis, initial colonization of the gastric mucosa,23 – 26 has been although with less T-cell infiltration. The gastritis pro- shown to be an active immunogen in protecting mice duced by H. felis infection was similar to that observed against H. felis infection.7 – 10 Approximately 50% of in humans infected with H. pylori in terms of infiltration rUre-immunized mice that were subsequently challenged of CD4/ and CD8/ lymphocytes, loss of parietal cells, with H. felis remained uninfected during the year thereaf- and hyperplasia of the surface epithelium.27 – 29 However, ter and had no significant gastritis. Challenge with H. the infiltration of T cells was deeply mucosal rather than felis increased lymphocyte counts in the gastric antrum, surface localized. This difference may be related to the which is the location in the stomach where the greatest panmucosal colonization by H. felis compared with the H. felis colonization occurs in the absence of immuniza- surface epithelial colonization by H. pylori.3,5,13,30 How- tion.3 – 6 After immunization, infiltration of lymphocytes ever, it may be a characteristic feature of murine gastritis, into the antrum probably represents a desirable, vaccine- because a similar deep mucosal to submucosal pattern is induced response to infection.4,10 However, antral gastri- present in mice with non–Helicobacter-related gastritis.31 tis did not resolve over time, and a marked lymphocytic Epithelial changes in the corpus consisted primarily of gastritis developed in the corpus during the 10 weeks parietal cell loss and hyperplasia of the surface epithelium after challenge in Ç15% of the mice and persisted during (foveolar hyperplasia), but microabscesses and cystic the subsequent year. By the end of the year-long study, glands were also observed. Similar changes were also a similar gastritis also developed in roughly the same found in chronically H. felis–infected mice from this as / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 9. 1126 ERMAK ET AL. GASTROENTEROLOGY Vol. 113, No. 4 pus and in preventing epithelial changes in the corpus to a level comparable with that found in controls. Likewise, CD4/ and CD8/ cell counts were significantly reduced in the antrum. The few cells still remaining in the gastric mucosa of rUre-immunized mice after treatment may have been present because the antimicrobial regimen was initiated 2 weeks after challenge (a time when an immune response against the organism was already established), and by the end of the study, the infiltration had not completely resolved. Increased CD8/ cells in the gastric mucosa may be related to the presence of residual bacteria at the apical surface or within epithelial cells. Gastric CD8/ cells per- sisted after challenge, indicating that the stimulus re- mained in the gastric tissue over time, but were reduced Figure 7. T-cell infiltration in protected vs. unprotected rUre-immu- after antimicrobial treatment. Intestinal IELs are believed nized H. felis –challenged mice. Unprotected mice were available at to play a role in immune surveillance against epithelial 10 (n Å 4), 17 (n Å 4), and 57 (n Å 2) weeks after challenge. injury by recognizing abnormal peptides derived from CD4/ cell and CD8/ gastritis did not develop in the corpus of any infectious agents and expressed on epithelial cells.36 Per- unprotected mice (significant differences from protected mice were not achieved). P values compare T-cell populations in Not Protected haps gastric IELs function in a similar manner. vs. Protected groups. Protected mice were defined as having a urease Gastritis in the corpus did not appear to be the conse- assay optical density value at 550 nm within 2 SD of the value in quence of autoantibody production against parietal cells. control mice (õ0.2); Not Protected mice were defined as having a urease assay optical density value at 550 nm of ú0.5 or a bacterial No reactivity was observed in this study or another study score of ¢3. in which mice were immunized with either urease A or B subunits.37 The T-cell response was not limited to the corpus but included the antrum as well. Furthermore, well as other studies.3,32 Mucous cell metaplasia in H. CD8/ IELs do not appear to be a component of murine felis–challenged mice occurred in the surface epithelium autoimmune gastritis, where anti–parietal cell autoanti- of the corpus and appeared distinct from H. pylori–associ- bodies can be detected in sera.31 ated metaplasia in humans, where the distribution is It is not clear why corresponding levels of gastritis characteristically antral and mucous cells can undergo were not observed in unprotected rUre-immunized mice complete metaplasia into intestinal-type goblet cells.33 if residual bacteria per se are responsible for gastritis. In Gastritis in rUre-immunized mice was characterized these mice, bacterial infection was high, but a significant by the presence of both CD4/ and CD8/ T cells, al- lymphocytic infiltration was not produced. One factor in though initially (at 2 weeks) it involved primarily CD4/ murine inflammation that may account in part for this cells. In other studies, these CD4/ cells have been shown to primarily express a,b-TCR.10 T cells expressed the mucosal homing integrin b7 , which is present on a4b7/ Table 2. Relative Proportion of T Cells in Gastric Mucosa of lymphocytes homing to mucosal tissues.18,19,22,34 Virtu- a Subset of H. felis –Challenged Mice ally all CD8/ cells represented gastric aE-integrin–posi- rUre / LT LT tive IELs. The aE-integrin is present on intestinal IELs T-cell antigen Antrum Corpus Antrum Corpus as part of aEb7 , the ligand for E-cadherin on epithelial cells, but not on peripheral T cells.18,19,22 A subset of CD4 (RM4-5) 181 { 19 252 { 37 104 { 16 77 { 5 CD8a (53-6.7) 96 { 17 197 { 22 õ1 õ1 gastric CD8/ cells also appeared to express Vb6 , which b7-Integrin (M293) 269 { 33 406 { 54 85 { 16 44 { 8 is found at relatively high frequency on intestinal IELs aE-Integrin (M290)a 147 { 21 271 { 31 44 { 7 15 { 4 bearing homodimeric CD8a chains.35 Together, these Vb6 (RR4-7) 49 { 15 91 { 30 õ2 õ2 data support the hypothesis that gastric CD8/ cells rep- NOTE. 8 rUre-immunized mice with the highest CD4/ cell and CD8/ resent a population of cells in the stomach corresponding cell counts and 8 LT-treated mice with the highest CD4/ cell counts to or derived from intestinal IELs. were analyzed for expression of b 7 -integrin, aE-integrin, and Vb6 . T- ˜ cell counts made in serial sections. Triple antimicrobial treatment was effective in reduc- a The T-cell region of the spleen contained only scattered aE-integrin– ing lymphocytic infiltration in both the antrum and cor- positive cells, which represented õ10% of CD8/ cells. / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
  • 10. October 1997 ORAL IMMUNIZATION WITH RECOMBINANT UREASE 1127 Figure 8. Expression of aE-inte- grin on purified gastric CD8/ IELs as determined by two-color flow cytometry. More than 90% of gastric CD8/ IELs expressed aE-integrin. (A ) Gastric and (B ) intestinal IELs were isolated by incubating tissue in Ca2/, Mg2/ –free EDTA-HBSS me- dium and purified by Percoll gra- dient centrifugation. IELs were pooled from 10 rUre- plus CT- immunized mice. observation is host responsiveness. It has been shown that A comparison of H. pylori vaccines in murine H. felis some mouse strains, such as BALB/c, develop low-grade models indicates that rUre is among the most efficacious inflammation in response to H. felis, whereas other single Helicobacter protein examined to date.8,39 Oral im- strains, such as C57BL/6, develop high-grade inflamma- munization with rUre produces a long-lasting reduction tion.38 In our experimental model using outbred mice, in H. felis organisms, but sterilizing immunity may not bacterial density in itself does not appear to determine be achieved under these experimental conditions, re- the inflammatory response. In addition to host factors, sulting in the development of a persistent T-cell gastritis bacterial virulence factors, which are poorly understood in Ç10%–20% of cases. This response does not seem in H. felis, may also contribute to inflammation. In the to be unique to rUre, because a mixture of Helicobacter case of a chronic infection such as H. felis, genetic diversi- antigens may also produce a persistent inflammatory re- fication or selection may occur over time, leading to sponse.8 Further studies are under way to determine different levels of inflammation. whether these phenomena occur in nonmurine hosts and to develop vaccines capable of eliminating bacteria from the gastric mucosa. Table 3. Effect of Antimicrobial Triple Therapy on Histopathology in the Antrum and Corpus of rUre- References Immunized, H. felis –Challenged Mice 1. Blaser MJ. Helicobacter pylori and the pathogenesis of gastrodu- Parameter rUre / CT rUre / CT / Rx Pa odenal inflammation. J Infect Dis 1990;161:626–633. 2. Cover TL, Blaser MJ. Helicobacter pylori and gastroduodenal dis- Antrum ease. Annu Rev Med 1992;43:135–145. Median gastritis score 2 (1–3) 1 (1–2) 3. Fox JG, Lee A, Otto G, Taylor NS, Murphy JC. Helicobacter felis (range) (n Å 20) (n Å 20) õ0.01 gastritis in gnotobiotic rats: an animal model of Helicobacter Median epithelial 1 (0–3) 0 (0–1) pylori gastritis. Infect Immun 1991;59:785–791. score (range) (n Å 20) (n Å 20) õ0.01 4. Fox JG, Blanco M, Murphy JC, Taylor NS, Lee A, Kabok Z, Pappo CD4/ cells 130 { 45 50 { 26 J. Local and systemic immune responses in murine Helicobacter (mean { SD) (n Å 11) (n Å 9) õ0.01 felis active chronic gastritis. Infect Immun 1993;61:2309– CD8/ cells 78 { 65 3{3 2315. (mean { SD) (n Å 11) (n Å 9) õ0.01 5. Lee A, Fox JG, Otto G, Murphy J. A small animal model of human Corpus Helicobacter pylori active chronic gastritis. Gastroenterology Median gastritis score 2 (0–3) 0 (0–1) 1990;99:1315–1323. (range) (n Å 20) (n Å 20) õ0.01 Median epithelial 1 (0–3) 0 (0–1) 6. Chen M, Lee A, Hazell SL. Immunization against Helicobacter score (range) (n Å 20) (n Å 20) 0.04 infection in a mouse/Helicobacter felis model. Lancet 1992; CD4/ cells 122 { 116 54 { 29 339:1120–1121. (mean { SD) (n Å 11) (n Å 9) NS ` 7. Michetti P, Corthesy–Theulaz I, Davin C, Haas R, Vaney A–C, CD8/ cells 135 { 228 7{3 Heitz M, Bille J, Kraehenbuhl J–P, Saraga E, Blum AL. Immuniza- (mean { SD) (n Å 11) (n Å 9) NSb tion of BALB/c mice against Helicobacter felis infection with Heli- cobacter pylori urease. Gastroenterology 1994;107:1002– a Wilcoxon’s rank sum test. 1011. b P õ 0.07. 8. Ferrero RL, Thiberge JM, Huerre M, Labigne A. Recombinant anti- / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro
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Science 1995;267:1655–1658. system. Eur J Immunol 1991;21:2591–2597. 23. Tsuda M, Karita M, Morshed MG, Okita K, Nakazawa T. A urease- negative mutant of Helicobacter pylori constructed by allelic ex- Received November 22, 1996. Accepted July 1, 1997. change mutagenesis lacks the ability to colonize the nude mouse Address requests for reprints to: Thomas H. Ermak, Ph.D., OraVax, stomach. Infect Immun 1994;62:3586–3589. Inc., 38 Sidney Street, Cambridge, Massachusetts 02139. Fax: 24. Eaton, KA, Krakowka S. Effect of gastric pH on urease-dependent (617) 494-0927. colonization of gnotobiotic piglets by Helicobacter pylori. Infect The authors thank Timothy Tibbitts, Jennifer Bakios, Kathleen Immun 1994;62:3604–3607. Georgakopoulos, and Heather Gray for excellent technical assis- 25. Eaton, KA, Morgan DR, Krakowka S. Motility as a factor in the tance. / 5e21$$0043 09-09-97 13:00:34 gasas WBS-Gastro