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308 Protective Anti-Helicobacter Immunity Is Induced with Aluminum Hydroxide or Complete Freund’s Adjuvant by Systemic Immunization Judith M. Gottwein,1 Thomas G. Blanchard,2 Oleg S. Targoni,1 Julia C. Eisenberg,1 Brandon M. Zagorski,1 Raymond W. Redline,1 John G. Nedrud,1 Magdalena Tary-Lehmann,1 Paul V. Lehmann,1,a and Steven J. Czinn2 Departments of 1Pathology and 2Pediatrics, Case Western Reserve University, Cleveland, Ohio To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5– secreting, antigen-specific T cells, and immunization with antigen and complete Freund’s adjuvant induced interferon-g–secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient mMT mice (immunoglobulin-gene knockout mice), and CD4+ spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1⫺/⫺ recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine. Helicobacter pylori, one of the world’s most prevalent pathogens, is an extracellular bacterium that infects the gastric mucosa and plays an etiologic role in gastritis and peptic ulcer disease [1, 2]. It is generally believed that infection occurs primarily in young children. Therefore, a prophylactic vaccine administered to infants might prevent H. pylori infection and the long-term consequences that occur in adults. Vaccination strategies have focused primarily on orally and intranasally administered immunizations to induce mucosal immunity [3]. These immunizations require bacterial exotoxin adjuvants that are unsafe for use in humans. Systemic immunization has recently been reported as a possible means of inducing protective Received 5 January 2001; revised 16 April 2001; electronically published 10 July 2001. Presented in part: 100th annual meeting of the American Gastroenterology Association, Orlando, Florida, April 1999 (abstract A695). All animal handling and experimentation was reviewed by, and performed according to the guidelines of, the Institutional Animal Use and Care Committee of Case Western Reserve University. The Case Western Reserve University animal facility is fully accredited by the American Association for Accreditation of Animal Care. Financial support: National Institutes of Health (research grants DK46461 and AI-36359 to S.J.C., DK-48799 and AI-42635-01 to P.V.L., and AI-40701 to J.G.N.); National Multiple Sclerosis Society (grant 2470 A-1/ 2 to P.V.L.). J.M.G. is a fellow of the Studienstifung des Deutschen Volkes. a P.V.L. is president of Cellular Technologies, whose product was used in these studies to determine the number of cytokine-producing cells. Reprints or correspondence: Dr. Steven J. Czinn, Dept. of Pediatrics, Rainbow Babies & Children’s Hospital, 2101 Adelbert Rd., Cleveland, OH 44106 ([email protected]). The Journal of Infectious Diseases 2001; 184:308–14 䉷 2001 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2001/18403-0008$02.00 immunity against H. pylori in mice [4]. The authors promoted the idea that contribution of Th1-mediated responses, the response associated with H. pylori–related gastric pathology, are required to induce immunity [4]. This suggestion was based on immunoglobulin (Ig) G subclass analysis only; no direct measurement of cytokine profiles was performed [4, 5]. Because several reports have suggested that protection might be mediated by type 2 immunity [6, 7], we investigated this in a more direct fashion by use of parenteral immunization with adjuvants that yield highly polarized Th1 or Th2 responses, as measured by ELISPOT assay. In the present study, we investigated whether systemic immunization with the adjuvant aluminum hydroxide (AlOH) can induce protective immunity that is mediated by Th2 CD4⫹ T cells in the absence of antibody. AlOH-induced immunity would represent a significant advance for the development of an H. pylori vaccine, because AlOH is already approved for use in humans. Its safety, stability, and low cost could expedite development of a human H. pylori vaccine suited for large-scale application. Materials and Methods Mice. Mouse strains were purchased from Jackson Laboratories and were housed under specific-pathogen-free conditions in microisolator units. The mice used were either C57BL/6 or C57BL/ 6J-Rag1tm1Mom (rag1⫺/⫺), deficient in mature T and B lymphocytes, or C57BL/6-Igh-6tm1Cgn (mMT), deficient in mature B cells, neither of which can make antibodies. Bacteria. H. felis was isolated in our laboratory from a feline gastric biopsy sample [8]. H. pylori strain HpM6 was isolated from JID 2001;184 (1 August) H. pylori Immunity with a Human-Use Adjuvant a human gastric biopsy sample and was adapted in our laboratory to mice via long-term in vivo passage. HpM6 was determined to be cagA positive by polymerase chain reaction. Inoculation of C57BL/6 mice with HpM6 results in chronic infection that persists for 112 months in 100% of infected mice. Both Helicobacter species were identified on the basis of colony morphology, bacterial morphology, Gram stain, and the production of urease, catalase, and oxidase. Bacteria were grown on solid media (Columbia blood agar) and were resuspended in brucella broth. Immunization and challenge of mice. Helicobacter lysate, which we and others have demonstrated to be an effective experimental vaccine antigen by the oral route [6, 8–11], was generated in our laboratory, as described elsewhere [9]. Ovalbumin (OVA) was purchased from Sigma Chemical Co., AlOH (Imject Alum) was purchased from Pierce, and complete Freund’s adjuvant (CFA) was made by mixing Mycobacterium tuberculosis H37RA (Difco Laboratories) at 1 mg/mL into incomplete Freund’s adjuvant (Gibco BRL). Antigens in aqueous solution were mixed 1:1 with the adjuvant, and 100 mg of antigen in 100 mL of emulsion was injected intraperitoneally into each mouse on day 0. On day 28, bacterial challenge with 1 ⫻ 10 7 cfu of bacteria in 0.5 mL of nutrient broth was given by gastric intubation with flexible tubing on an 18-gauge needle. Bacterial numbers for challenge were determined by optical density at 450 nm by use of a previously established growth curve. Although H. felis is difficult to grow as isolated colonies, we use a standard absorbance value that is based on the H. pylori growth curve for both organisms, as described elsewhere [9]. Determination of vaccine efficacy. Twenty-eight days after challenge, the mice were examined for infection by Helicobacter organisms by silver staining of tissue (Steiner stain) [9, 12]. The animals were killed by CO2 asphyxiation, and a narrow strip of tissue was surgically removed from the greater curvature of the stomach, from the duodenum to the gastric cardia. Tissues were fixed in 10% buffered formalin and were processed for histologic examination at the University Hospitals of Cleveland Histology Laboratory. Several sections of each mouse sample were stained by the Steiner method, to facilitate the identification of H. pylori and H. felis on the basis of bacterial location and morphology. The mice were considered to be protected if there was a complete absence of detectable Helicobacter organisms in silver-stained sections. Additionally, cultures were performed on gastric biopsy samples from H. pylori–challenged mice to confirm the presence or absence of bacteria. Two biopsy samples (2 ⫻ 2 mm) from mice challenged with H. pylori were surgically removed from the gastric antrum and were homogenized in 200 mL of Columbia broth. Homogenates were plated on Columbia agar supplemented with 7% horse blood in 100mL aliquots. After 96 h in microaerophilic conditions, confirmation of protection was determined by the absence of any culturable bacteria from immunized mice. When present, bacteria were confirmed as H. pylori on the basis of colony morphology, Gram stain, and the production of urease, catalase, and oxidase. Cultures for H. felis were not performed because culture is less reliable, and H. felis tends to grow as a bacterial lawn instead of as isolated colonies. Evaluation of pathology. Longitudinal sections of the greater curvature of the mouse stomach were evaluated for overall intensity of inflammation, as described elsewhere [9, 13]. Sections included the entire length of both the antral and fundic glandular mucosa. Antral inflammation was graded on a 0–3 scale, and fundic in- 309 flammation on a 0–10 scale. A global score for each mouse was determined on the basis of the linear extent (focal, multifocal, patchy, or diffuse), depth (superficial and/or basal, panmucosal, or extending to submucosa or muscular layers), and character of the inflammatory infiltrate, as defined by the types of infiltrating cells and tissue architecture changes. Adoptive transfer. CD4⫹ T cells were purified with the mouse T cell CD4 subset column kit (R&D Systems). Ten million cells were transferred to each recipient mouse via injection into the tail vein. These numbers have been established and are customarily used in the field of autoimmune research [14] ELISPOT assay. Single-cell suspensions were prepared from the spleen, and 1 ⫻ 10 6 cells were plated per well in serum-free HL1 medium (BioWhittaker) supplemented with L-glutamine at 1 mM, with or without Helicobacter antigens added, at a final concentration of 5 mg/mL. These cultures were added to ELISPOT plates (ImmunoSpot; Cellular Technologies) that were precoated overnight with capture antibodies specific for interferon (IFN)–g or interleukin (IL)–5, R46-A2 (4 mg/mL), or TRFK5 (5 mg/mL), respectively, in PBS. The plates were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature and were washed 4 times with PBS before the beginning of the 24-h cell culture. Next, the cells were removed by washing, the detection antibody (XMG1.2-HRP at 1 mg/mL for IFN-g or TRFK4 at 4 mg/mL for IL-5) was added, and the plates were incubated overnight. For IL5, anti–IgG2a-HRP (Zymed) was added and was incubated for 2 h. The plate-bound final antibody then was visualized by adding 3-amino-9-ethyl-carbazole. To evaluate the results, we used a Series 1 ImmunoSpot Image Analyzer (Cellular Technologies). Statistical analysis. Statistical significance between experimental groups for ELISPOT analysis was determined by analysis of variance. The presence or absence of experimental infection after challenge of immunized mice was evaluated for significance by Fisher’s exact test. Results Murine models of Helicobacter infection and immunity have used both the human pathogen itself or a related organism isolated from cats, H. felis [15]. Unlike H. pylori, H. felis induces inflammatory lesions in the gastric mucosa, reminiscent of human gastritis, when the mouse strain C57BL/6 is used [13, 16]. Additionally, H. felis appears to yield a heavier infection, as gauged by histology, in the gastric tissue of mice, and unlike H. pylori, which primarily resides at the antral-fundic junction, its colonization pattern includes most of the antrum and fundus. We systemically immunized C57BL/6 mice with H. felis or H. pylori antigens emulsified in either AlOH or CFA; we have recently shown that these adjuvants can be used to induce polarized type 2 and type 1 immunity, respectively [17]. Fourteen days later, the spleen cells of these mice were tested for Helicobacter antigen–specific recall response by measuring a type 1 and type 2 cytokine (IFN-g and IL-5, respectively) by ELISPOT assay. Cells from mice immunized with antigen in AlOH produced IL-5 in the virtual absence of IFN-g (figure 1A and 1C). The number of IL-5–producing cells was signifi- 310 Gottwein et al. Figure 1. Induction of systemic, adjuvant-guided type 1 and type 2 immunity to Helicobacter antigens. Mice were injected with H. pylori lysate in aluminum hydroxide (AlOH) (A) or complete Freund’s adjuvant (CFA) (B), and 14 days later their spleen cells were tested by ELISPOT assays for interferon (IFN)–g (⽧) or interleukin (IL)–5 (䡬) production in the presence of medium or H. pylori antigen, as indicated. Each symbol represents the mean no. (of triplicate wells) of spot-forming cells per million spleen cells per individual mouse. AlOH induced significantly more IL-5–producing cells than IFN-g–producing cells (P ! .0001), whereas CFA induced significantly more IFN-g–producing cells than IL-5–producing cells (P ! .0001). Mice immunized with H. felis antigen in AlOH (C) or CFA (D) were tested under identical conditions. Again, AlOH induced significantly more IL-5–producing cells than IFN-g–producing cells (P ! .003 ), whereas CFA induced significantly more IFN-g–producing cells than IL-5–producing cells (P ! .0001). The data are from 1 experiment, representative of the 3 experiments performed. Statistical significance of differences between experimental groups was determined by analysis of variance. cantly greater than that of antigen-specific IFN-g–producing cells for both H. pylori antigen immunization (P ! .0001) and H. felis antigen immunization (P ! .003 ). The reverse cytokine profile was seen after immunization with antigen and CFA (figure 1B and 1D). The number of IFN-g–producing cells induced by the recall response was significantly greater than that of IL5–producing cells (P ! .0001 for both H. pylori and H. felis antigens). Additionally, serum samples from the CFA-immunized mice contained high titers of specific IgG2a antibodies, whereas AlOH-immunized mice had an IgG1 antibody response that prevailed over IgG2a (data not shown). Thus, on the basis of both the relative numbers of cells producing the respective cytokines and the antibody isotype profiles of these immune responses, immunization with AlOH induced polarized JID 2001;184 (1 August) type 2 immunity, whereas immunization with CFA triggered type 1 immunity against the coinjected Helicobacter antigens. We then tested whether the type 1 or type 2 immunity induced by systemic immunization with Helicobacter antigens would confer protection against Helicobacter infection of the gastric mucosa. C57BL/6 mice were immunized with H. pylori or H. felis antigens emulsified with AlOH or CFA; control mice were injected with these adjuvants containing an irrelevant control protein, OVA, or remained unimmunized. Mice were challenged orally with 1 ⫻ 10 7 bacteria of the respective Helicobacter species on day 28, and 28 days later, the bacteria load was assessed by direct visualization of silver-stained histologic sections of the gastric mucosa (Steiner staining). The presence or absence of Helicobacter organisms was confirmed by culture (see Materials and Methods). These studies of fully immunocompetent, wild-type mice demonstrated that systemic immunization with either adjuvant afforded profound protection, as determined by the complete absence of Helicobacter organisms in silverstained histologic sections (table 1). Although 12 (86%) of 14 control mice became infected with H. pylori, 7 (88%) of 8 mice injected with H. pylori:AlOH were free of infection. This protection was significant for both AlOH (P p .005) and CFA (P p .049) adjuvants. Results with H. felis, which causes a more aggressive infection in mice than does H. pylori, were even more striking. Ninetysix percent (28 of 29) of the control mice became infected and exhibited a high bacteria load, averaging 86 infected glands per Table 1. CD4⫹ T cell–mediated protection against Helicobacter infection induced by systemic vaccination in mice. C57BL/6 a strain WT mMT Experimental group Immunization Challenge No. d infected A B C D E F G H I J K Hp:AlOH Hp:CFA OVA:AlOH OVA:CFA Hf:AlOH Hf:CFA OVA:AlOH OVA:CFA None Hf:AlOH OVA:AlOH Hp Hp Hp Hp Hf Hf Hf Hf Hf Hf Hf 1/8 2/8 7/8 5/6 0/5 5/21 5/5 16/17 7/7 1/8 8/8 b c e P .005 .049 .004 !.001 !.001 NOTE. AlOH, aluminum hydroxide; CFA, complete Freund’s adjuvant; Hf, Helicobacter felis antigen; Hp, Helicobacter pylori antigen; OVA, ovalbumin; WT, wild type. a Strains included C57BL/6 (WT) mice (groups A–I) and immunoglobulingene knockout (mMT) mice on the same background (groups J and K). b A total of 100 mg of Hp, Hf, or OVA, with AlOH or CFA, as specified. c Mice were challenged orally with 1 ⫻ 107 cfu of the respective Helicobacter strain. d Protection is defined as the complete absence of detectable bacteria. The bacteria load in the gastric mucosa was assessed in all groups of mice 4 weeks after infectious challenge, by counting silver stain–positive bacteria and scoring as positive glands per longitudinal section. For experiments performed with Hp, cultures also were performed on biopsy samples, to confirm the absence of bacteria in protected mice. e The presence or absence of experimental infection after challenge of immunized mice was evaluated by Fisher’s exact test for significance vs. corresponding OVA-immunized mice. JID 2001;184 (1 August) H. pylori Immunity with a Human-Use Adjuvant stomach section. No bacteria could be found in any of the 5 H. felis:AlOH–vaccinated mice. Immunizations with Helicobacter antigens in CFA were also protective (5 of 21 mice were infected). This level of protection was significant for both AlOH (P p .004) and CFA (P ! .001) adjuvants and is similar to what we and others have observed when the most effective known mucosal vaccination strategies that require cholera toxin as the adjuvant are used (reviewed in [3]). Figure 2 demonstrates the variation in bacteria load observed in silver-stained gastric sections among groups of mice. No bacteria were observed in H. felis:AlOH–vaccinated mice, versus large numbers of infected glands in sham-immunized mice and small numbers of infected glands in H. felis:CFA–vaccinated mice. Systemic induction of type 1 or type 2 immunity may, therefore, be a viable alternative to mucosal immunization against 311 Helicobacter infection. Examination of hematoxylin-eosin– stained sections from these mice revealed that immunization of mice with H. felis antigens in combination with either AlOH or CFA each resulted in equivalent levels of gastric inflammation at both the antrum and fundic mucosa. The degree and character of the inflammation were not appreciably different from those previously observed by us for orally administered immunization with Helicobacter antigen and cholera toxin adjuvant [9]. Mice immunized with Helicobacter antigens had average fundic inflammation of 7.2 Ⳳ 0.9 and 7.4 Ⳳ 0.7 when AlOH or CFA was used, respectively. Antral inflammation was 2.0 Ⳳ 0 and 2.1 Ⳳ 0.6 for AlOH and CFA, respectively. In all cases, mice previously immunized with Helicobacter antigen had a greater degree of inflammation after challenge than did mice immunized with OVA. This is consistent with the post- Figure 2. Histologic illustration that systemic immunization prevents Helicobacter infection. Sections of gastric mucosa from immunized mice challenged with H. felis are shown. The most heavily colonized sections are shown for mice immunized with H. felis sonicate plus aluminum hydroxide (AlOH; table 1, group E, no organisms visible) (A) and H. felis sonicate plus complete Freund’s adjuvant (CFA; table 1, group F) (B), and typical sections are shown for mice that were sham immunized with ovalbumin (OVA) plus AlOH (table 1, group G) (C) and OVA plus CFA (table 1, group H) (D). Arrows indicate an individual H. felis organism or a group of H. felis organisms in the tissue section (Steiner stain; original magnification, ⫻200). 312 Gottwein et al. immune gastritis observed in many laboratories and is most likely due to the development of anti-Helicobacter immune memory secondary to the immunization. We next began to investigate the immune mechanism that mediates anti-Helicobacter immunity after systemic immunization. Because CFA, like cholera toxin, is highly toxic and unsuitable for use in humans, we focused on the use of AlOH as an adjuvant. In these subsequent experiments, we used the murine–H. felis model, because this organism exhibits more pronounced inflammation in mice than does H. pylori and also results in a heavier infection that can readily be detected in histologic sections. We immunized congenic mMT mice (immunoglobulin-gene knockout mice) with H. felis antigen: AlOH, or with OVA:AlOH (table 1, groups J and K) to determine whether the presence of antibody is necessary for protection after parental immunization. All 8 of the OVA-control mMT mice became infected after challenge with H. felis, exhibiting a high bacteria load (n p 65 positive glands per longitudinal section of gastric mucosa). However, only one of the H. felis antigen:AlOH–injected mMT mice became infected (2 positive glands per longitudinal section of gastric mucosa), indicating significant protection (P ! .001 ). Inflammation in these mice was not appreciably different from that in the fully immunocompetent B6 mice, because immunization with H. felis antigen and AlOH resulted in postchallenge fundic inflammation of 7.8 Ⳳ 0.6 and antral inflammation of 2.38 Ⳳ 0.5. Immunization with OVA and AlOH resulted in average scores of 3.7 Ⳳ 1.5 in the fundus and 1.5 Ⳳ 0.5 in the antrum. This is consistent with our previous use of mMT mice for orally administered immunization [9]. Because these experiments suggested that antibodies are not JID 2001;184 (1 August) required for immunity after parenteral vaccination, we next performed an experiment to determine whether purified T cell populations from vaccinated mice can adoptively transfer protection. CD4⫹ T cells were purified by affinity columns from the spleens of C57BL/6 mice on day 28 after immunization with either H. felis antigen:AlOH, OVA:AlOH, H. felis antigen:CFA, or OVA: CFA. Ten million cells (194% CD4⫹ by flow cytometry) were injected into each congenic, immunodeficient rag1⫺/⫺ recipient mouse (table 2, groups L, M, P, and Q). The recipient mice were challenged 3 days later with 1 ⫻ 10 7 H. felis organisms, and 28 days later, the bacteria load of the gastric mucosa was measured by inspection of silver-stained histologic sections. All 3 rag1⫺/⫺ mice reconstituted with OVA:AlOH–primed CD4⫹ T cells became heavily infected, exhibiting an average of almost 30 positive glands per section. Similarly, all 8 rag1⫺/⫺ mice reconstituted with OVA:CFA–primed CD4⫹ T cells had average H. felis loads of 150 positive glands per section. In contrast, 6 (75%) of the 8 rag1⫺/⫺ mice that were grafted with H. felis antigen:AlOH–primed CD4⫹ T cells had undetectable levels of bacteria (table 2, group L). Furthermore, the 2 mice that did become infected had a markedly reduced number of total Helicobacter organisms, ∼4% of that observed in control mice (data not shown). Therefore, the gastric immunity induced by systemic immunization with H. felis antigen and AlOH was mediated by CD4⫹ T cells. Although this protection did not quite achieve statistical significance (P p .061 ), this was most likely because of the reduced size of the control group (table 2, group M), the result of accidental deaths after a cage flooded. Histologic inflammation of these mice after challenge with H. felis was similar to the inflammation observed for challenge of immunized mice. The mice receiving CD4⫹ T cells from Helicobacter-im- Table 2. Helicobacter immunity in rag1⫺/⫺ mice by adoptive transfer of Agprimed CD4⫹ T cells. C57BL/6 a strain WT WT WT WT WT WT WT WT CD4rrag1⫺/⫺ CD4rrag1⫺/⫺ CD4rrag1⫺/⫺ CD4rrag1⫺/⫺ Experimental group Immunization Challenge No. d infected L M N O P Q R S Hf:AlOH OVA:AlOH Hf:AlOH OVA:AlOH Hf:CFA OVA:CFA Hf:CFA OVA:CFA Hf Hf Hf Hf Hf Hf Hf Hf 2/8 f 3/3 5/15 7/7 7/7 8/8 3/10 8/9 b c e P .061 .023 1.0 .014 NOTE. AlOH, aluminum hydroxide; CFA, complete Freund’s adjuvant; Hf, Helicobacter felis antigen; OVA, ovalbumin; WT, wild type. a Strains included C57BL/6 (WT) mice (groups N, O, R, and S) and rag1⫺/⫺ mice on the same background (groups L, M, P, and Q). b A total of 100 mg of Hf or OVA with AlOH or CFA, as specified. Naive rag1⫺/⫺ mice were injected with 1 ⫻ 107 CD4⫹ T cells isolated from spleens of WT mice immunized with either Hf or OVA, in combination with either AlOH or CFA, and challenged with Hf 3 days later. c Mice were challenged orally with 1 ⫻ 107 cfu of H. felis. d Protection is defined as the complete absence of detectable bacteria. The bacteria load in the gastric mucosa was assessed in all groups of mice 4 weeks after infectious challenge by counting silver stain–positive bacteria and scoring as positive glands per longitudinal section. e The presence or absence of experimental infection after challenge of immunized mice was evaluated for significance vs. corresponding OVA-immunized mice by Fisher’s exact test. f The small size of the group of mice receiving CD4⫹ T cells from OVA:AlOH-immunized mice was the result of the premature deaths of some of the recipient mice by drowning when their cage was accidentally flooded. JID 2001;184 (1 August) H. pylori Immunity with a Human-Use Adjuvant munized mice had average fundic inflammation of 7.3 Ⳳ 0.9 and antral inflammation of 2.3 Ⳳ 0.5. Inflammation in mice receiving OVA-primed T cells was slightly lower, with fundic scores averaging 6.7 Ⳳ 0.6 and antral scores of 1.7 Ⳳ 0.6. Mice engrafted with H. felis antigen:CFA–primed CD4⫹ T cells and then challenged with H. felis became uniformly infected, with average bacteria loads of 90 infected glands per section (table 2). Apparently an equal number of CFA-primed Th1 cells was less effective in transferring protection than was the same number of AlOH-primed Th2 cells. This is consistent with the reduced efficacy in CFA-immunized animals, compared with AlOH-immunized animals, observed in our other experiment (table 1, groups B and F). Alternatively, in the absence of regulatory networks present in wild-type mice, polarized Th2 CD4⫹ T cells alone (but not polarized Th1 cells alone) may be sufficient to mediate reduction in bacteria loads in the gastric mucosa. Discussion In summary, we have demonstrated that systemic immunization that uses AlOH as an adjuvant can induce protective immunity against H. pylori and H. felis in mice that is mediated by CD4⫹ type 2 cells. These findings may have a direct impact on the development of a Helicobacter vaccine for humans, for several reasons. First, and perhaps most importantly, the present report strengthens the concept that systemic immunization with an adjuvant approved for use in humans may be a viable alternative to experimental orally administered immunization for the induction of mucosal immunity against H. pylori. In further support of this, in a separate series of experiments designed to investigate the efficacy of parental immunization against H. pylori in neonates, we successfully and reproducibly used subcutaneous immunization to obtain protective immunity (authors’ unpublished data). This concept, backed by a recent report by Guy et al. [4], introduces a new strategy for H. pylori vaccination that is contrary to long-held principles of mucosal immunity. Second, contrary to the findings of Guy et al. [4], which indicated a requirement for type 1 responses in Helicobacter immunity, our data demonstrate that type 2 responses are at least equal to type 1 responses and are perhaps more efficacious in their ability to confer protective immunity against Helicobacter infection. AlOH, the only adjuvant available for human use, is a type 2 adjuvant. Some recent reports have suggested a role for mucosally induced type 2 immunity in protecting hosts from Helicobacter, including orally administered immunizations with cholera toxin and other experimental adjuvants that have elicited type 2 or mixed type 2–type 1 responses [6, 7]. Recently, Escherichia coli heat-labile toxin was used as an adjuvant to parentally immunize mice against H. pylori [5]. The heat-labile toxin was found to significantly increase the serum IgG1:IgG2a ratio, a possible indicator of type 2 immunity. Our data now show, at the cellular level, that a human-use adjuvant, 313 AlOH, directs a polarized type 2 systemic immune response that confers protection from Helicobacter infection in mice. Presumably, local release of either type 1 or type 2 cytokines in the stomach after parenteral immunization may be effective in recruiting or activating the effector cells that eliminate the bacteria. As such, it is possible that experimental Th1 adjuvants, such as CpG oligonucleotides, may also eventually prove to be effective at inducing protective H. pylori immunity. However, AlOH is already approved for human use and is widely used in humans, and the present study is encouraging in that it demonstrates that AlOH-induced type 2 immunity can induce protective immunity in mice. Third, we found that the Helicobacter immunity induced by systemic immunization can be conveyed by CD4⫹ T cells and can occur in the absence of antibodies. The gastric mucosa must, therefore, be included in the trafficking pathway of systemically primed CD4⫹ T cells providing immune surveillance [18]. This is consistent with recent reports by us and others demonstrating that protective immunity against Helicobacter, induced by mucosal immunization, is also antibody independent [9, 11, 19]. Induction of IgA-independent protective immunity against a noninvasive gastrointestinal bacterium suggests a novel CD4⫹ T cell–mediated immune effector mechanism at the gastric mucosa. Fourth, we have shown here that the type 1 bias of C57BL/ 6 mice [20] can be overridden by the intrinsic type 2 polarizing effects of AlOH [21]. Because humans are also biased toward either type 1 or type 2 responses [22–24], this finding suggests that uniform success might be accomplished with minimal side effects even in the mixed human population. The primary candidate for preventive vaccination will be young children, because they typically become infected within the first several years of life. Newborns are type 2 biased; therefore, the induction of type 2 immunity will be even further facilitated. Our ongoing work with neonatal mice has confirmed this concept (authors’ unpublished data). In humans, ulcers and gastritis are associated with a type 1 IFN-g response to H. pylori in the gastric mucosa [25–27], whereas AlOH induces Th2-type responses. Although AlOH-containing vaccines can induce antigen-specific IgE responses [17], AlOH has been widely used for human immunizations, including the common diphtheriatetanus–pertussis vaccinations. The notion put forth in this report—that a systemic immunization with Helicobacter antigens in AlOH is suited to induce a degree of immune protection that has been accomplished previously only by the use of toxic mucosal adjuvants or proprietary experimental adjuvants—should provide a novel rationale for, and expedite the development of, an H. pylori vaccine. References 1. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983; 1:1273–5. 2. NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. JAMA 1994; 272:65–9. 3. Blanchard TG, Czinn SJ, Nedrud JG. 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