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. Author manuscript; available in PMC: 2012 Mar 3.
Published in final edited form as: Vaccine. 2011 Jan 12;29(11):2085–2091. doi: 10.1016/j.vaccine.2010.12.130

HelicoVax: Epitope-based therapeutic H. pylori vaccination in a mouse model

Steven F Moss 1,*, Leonard Moise 2,3, Dong Soo Lee 1, Woojin Kim 1, Songhua Zhang 1, Jinhee Lee 4, Arlin B Rogers 5, William Martin 2, Anne S De Groot 2,3,6
PMCID: PMC3046230  NIHMSID: NIHMS268971  PMID: 21236233

Abstract

Helicobacter pylori is the leading cause of gastritis, peptic ulcer disease and gastric adenocarcinoma and lymphoma in humans. Due to the decreasing efficacy of anti-H. pylori antibiotic therapy in clinical practice, there is renewed interest in the development of anti-H. pylori vaccines. In this study an in silico-based approach was utilized to develop a multi-epitope DNA-prime/peptide-boost immunization strategy using informatics tools. The efficacy of this construct was then assessed as a therapeutic vaccine in a mouse model of gastric cancer induced by chronic H. pylori infection. The multi-epitope vaccine administered intranasally induced a broad immune response as determined by interferon-gamma production in ELISpot assays. This was associated with a significant reduction in H. pylori colonization compared with mice immunized with the same vaccine intramuscularly, given an empty plasmid, or given a whole H. pylori lysate intranasally as the immunogen. Total scores of gastric histological changes were not significantly different among the 4 experimental groups. These results suggest that further development of an epitope-based mucosal vaccine may be beneficial in eradicating H. pylori and reducing the burden of the associated gastric diseases in humans.

Introduction

Helicobacter pylori represents a significant public health challenge since it causes chronic gastritis and significantly increases the risk of developing peptic ulcer disease and gastric cancer [1]. H. pylori infection is highly prevalent, particularly in developing countries. The strong epidemiological linkage between H. pylori and distal (non-cardia) cancer led the World Health Organization's International Agency for Cancer Research branch to classify H. pylori as a definite (type I) carcinogen in 1994 [2], a conclusion confirmed by the same agency last year [3].

The co-evolution of H. pylori with human beings over tens of thousands of years and the inverse relationship between H. pylori and certain conditions, such as gastroesophageal reflux disease, has led to the suggestion that persistent H. pylori infection may provide some benefit to humanity, especially in the developed world [4]. Nevertheless, for most infected individuals the balance of evidence supports eradicating H. pylori to prevent gastritis, ulcer disease and gastric cancer, especially where H. pylori and gastric cancer are very common, such as in the Asian-Pacific region [5].

Current treatments to eradicate H. pylori involve the use of multiple antibiotics in combination with acid suppression medication over 1-2 weeks, an approach that has changed little over the last 3 decades since H. pylori was first identified [1]. Because of antibiotic resistance (especially to metronidazole and clarithromycin), H. pylori is more difficult to eradicate now than it had been formerly [6] leading to renewed interest in developing alternative therapeutic strategies such as vaccines [7]. Vaccination against H. pylori may be a particularly attractive choice as the most practical way to avoid H. pylori-associated gastric cancer in the developing world [8].

In humans, H. pylori stimulates an intense gastric inflammatory response that persists over the course of infection that typically lasts many decades. Animal models have established that the pathogenesis of gastric diseases induced by H. pylori is related to the Th1-skewed T-cell response and that humoral immunity does not provide protection against infection. Despite the generation of robust cellular and humoral immune responses, spontaneous eradication of the organism is extremely rare in humans [1], likely related to several properties of the bacterium that help it evade natural immune responses and allow persistence [4]. While normal immune mechanisms fail to eradicate H. pylori, some prophylactic and therapeutic vaccines strategies using a wide variety of H. pylori antigens have been reported to successfully eradicate H. pylori from mice and in a few studies in human volunteers [7]. Nevertheless the correlates of gastric immunity to H. pylori are not well defined and the optimal choice of antigen and best delivery method for further clinical development remain to be determined.

In order to advance our H. pylori vaccine strategy we used a gene-to-vaccine approach, incorporating multiple epitopes in a DNA-prime/peptide boost approach as previously described [9], using informatics tools that have been successfully applied to AIDS, TB, smallpox and tularemia vaccine development [9 - 13]. We present our initial findings on the efficacy of this construct as a therapeutic vaccine in a mouse model of gastric cancer induced by chronic H. pylori infection. Our results suggest that a broad-spectrum epitope-driven immune response against H. pylori antigens can lead to successful eradication of the organism.

Methods

Mice, H. pylori infection and overall experimental plan

A total of 80 male and female C57BL/6 mice deficient in the CDKN1B gene encoding p27 were used in this study. Because female heterozygous CDKN1B-deficient mice are sterile, the mice for this study were derived from breeding male homozygous deficient to female heterozygotes and then identifying homozygous deficient pups by genotyping. Heterozygote founders of this colony had been originally purchased from Jackson laboratories (Jackson Labs, Bar Harbor, ME). All studies were performed in full compliance with the standards of the Rhode Island Hospital Institutional Animal Care and Use Committee and in accordance with NIH publications entitled “Principles for Use of Animals” and “Guide for the Care and Use of Laboratory Animals.”

At 6 weeks of age mice were infected with 109 H. pylori strain SS1 in 0.1 mL PBS by gavage on 3 occasions over 1 week using a balled animal feeding needle as previously described [14].

There were 4 experimental groups (20 mice per group); the experimental design is shown in Figure 1. At weeks 11-15 of age, mice were given doses of a H. pylori SS1 lysate, an empty plasmid (“pVAX control”) or a DNA based vaccine intramuscularly or intranasally. At ages 17 and 18 weeks mice were given a boost of intranasal peptides formulated in liposomes (or empty liposome as a control).

Figure 1.

Figure 1

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Outline of study design.

All mice were maintained in micro-isolator cages to reduce inter-cage Helicobacter infection and allowed access to Harlan Teklad Global Diet 2018 (Harlan, Indianapolis, IN) and water ad libitum, in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of Rhode Island Hospital. At 51 weeks of age (45 weeks after H. pylori infection) mice were euthanized by cervical dislocation under isoflurane anesthesia for bacteriological, immunological and histological analyses.

Immunizations

A lysate of H. pylori strain SS1 was prepared by French press and clarified by centrifugation as described previously [15]. 200 microgram of H. pylori lysate with 10 microgram E. coli heat-labile LT (R192G) adjuvant in 20 microliters PBS was administered intranasally once weekly for 5 weeks. [16].

In the group receiving pVAX control intranasally, 100 micrograms of an empty plasmid was given with 10 microgram LT (R192G) adjuvant in 20 microliters PBS on 3 occasions over 5 weeks (weeks 11,13 and15), and 20 microliters PBS only was given intranasally on two occasions (weeks 12 and 14).

In the group receiving DNA vaccine intramuscularly, 100 micrograms of DNA in 0.1 milliliter PBS was given into the flank on 3 occasions over 5 weeks (weeks 11,13 and 15) and 20 microliters PBS only was given intranasally on two occasions (weeks 12 and 14).

In the group receiving DNA vaccine intranasally, 100 micrograms of DNA was given with 10 microgram LT (R192G) adjuvant in 20 microliters PBS on 3 occasions over 5 weeks (weeks 11,13 and 15) and 20 microliters PBS only was given intranasally on two occasions (weeks 12 and 14).

To boost responses in the two DNA-based vaccine arms, a 10 microliter volume of liposomal peptide vaccines (50 microgram) with 20 microgram immunostimulatory CpG oligonucleotides and 10 microgram LT (R192G) were given intranasally at week 17 and at week 19. All peptides were formulated together in a single preparation so that all the mice treated with peptides receive the identical peptide combination. The other two groups of mice (H. pylori lysate, pVax control) were given empty liposomes instead, at the same timepoints.

Multi-epitope DNA vaccine engineering

Epitope sequences were concatenated to form two multi-epitope genes (termed HelicoVax A and HelicoVax B), each containing 25 HLA Class II epitopes that were identified by immunoinformatics methods, as described previously [17]. The detailed final composition and epitope order are listed in Supplemental Table 1 and Supplemental Table 2. All mice in the DNA vaccine groups received both HelicoVax A and HelicoVax B. Initially, epitopes were assembled in a random sequence. To avoid creation of novel epitopes at epitope junctions, an algorithm which iteratively re-orders epitopes to reduce junctional immunogenicity (VaccineCAD) was used to optimize epitope order [10]. In addition, Gly-Pro-Gly-Pro-Gly spacer sequences were engineered between some epitopes to optimize epitope processing, where re-ordering by VaccineCAD did not sufficiently reduce potential junctional immunogenicity [18].

A Kozak sequence was engineered upstream of the coding sequence for efficient translation initiation. To target the immunogens to the Class II processing pathway, the tissue plasminogen activator (tPA) leader sequence (MQMSPALTCLVLGLALVFGEGSA) was placed upstream of epitope sequences to direct translation products to the secretory pathway. A histidine tag was incorporated downstream of the epitope sequences followed by two stop codons. Genes were synthesized by GeneArt (Burlingame, CA) and subcloned at pre-determined flanking restriction sites into pVAX1 (Invitrogen, Carlsbad, CA), a DNA vaccine vector that accommodates FDA recommendations for construction of plasmid DNA vaccines [19]. High purity plasmids for immunizations were prepared by PureSyn, Inc. (Malvern, PA) at pre-clinical grade.

Peptide vaccine preparation

Peptides were manufactured using 9-fluoronylmethoxycarbonyl (Fmoc) chemistry by New England Peptide (Gardner, MA). Master batch records indicate that peptides were purified to >80% as ascertained by analytical reversed phase HPLC and peptide mass was confirmed by MALDI-TOF mass spectrometry.

The constituent peptides of the DNA vaccine were formulated in liposomes with immunostimulatory CpG oligodeoxynucleotide (ODN) 1555 (5′-GCTAGACGTTAGCGT-3′; Integrated DNA Technologies, Coralville, IA) [20] and heat-labile enterotoxin, LT (R192G), kindly provided by Dr. John Clements (Tulane University). Sterically stable cationic liposomes were prepared from three lipid components: 11.9 μmol dioleylphosphatidylethanolamine, 7.9 μmol dimethylaminoethanecarbamol-cholesterol, and 1.1 μmol polyethylene glycol 2000-phosphatidyl-ethanolamine (Avanti Polar Lipids, Alabaster, AL). The lipids were mixed, dried in a rotary evaporator and re-suspended in PBS to make empty multi-lamellar vesicles. These vesicles were then sonicated for 2 min to convert them into unilamellar liposomes. Unilamellar liposomes (10 μmol) were mixed with CpG ODN (400 μg) and peptides (1 mg), flash frozen and freeze-dried overnight. To encapsulate CpG ODN and peptides in liposomes, the resulting powder was re-suspended with sterile distilled water and vortexed for 15 seconds every five minutes for 30 minutes at room temperature. PBS and LT (R192G) were added to yield the final required vaccine volume and LT dose, as described above. Liposome formulations were stored at 4°C until use.

H. pylori quantification

H. pylori infection was quantified by real-time PCR of the SSA gene of H. pylori, normalized for mouse stomach GAPDH expression, as previously described [14].

Gastric histology

Gastric histology was evaluated without knowledge of experimental group in H&E-stained sections and scored for epithelial and inflammatory cell parameters using the criteria and grading system of Rogers et al [21].

ELISpot assay

The frequency of epitope-specific splenocytes was determined by IFN-gamma ELISpot assay using the Mabtech IFN-gamma ELISpot Kit according to the manufacturer's protocol (Mariemont, OH). Briefly, spleens were harvested from groups of control and vaccinated mice and mascerated to produce single cell splenocyte suspensions in RPMI-10% fetal bovine serum-1% penicillin/streptomycin-1% L-glutamine-0.1% BME at a concentration of 3 × 106 cells/ml. Cells were transferred at 3×105/well to ELISpot plates pre-coated with anti-murine IFN-gamma by the manufacturer and stimulated with 50 individual and 6 pools of 5-10 peptides at 10 μg/ml in triplicate wells. Pools 1 to 3 correspond to the epitopes contained in DNA vaccine construct A (HelicoVaxA) and pools 4 to 6 correspond to epitopes present in DNA vaccine construct B (HelicoVaxB). Positive controls included cells stimulated with Con A (2 μg/ml) or SS1 lysate (10 μg/ml); no-peptide stimulation served as a negative control. ELISpot plates were incubated at 37°C, 5% CO2 for 2 days, washed, incubated with a secondary HRP labeled anti-IFN-gamma antibody and developed by addition of TMB substrate. Spot counts were determined by Zellnet, Inc. using a Zeiss ELISpot plate reader. Results were recorded as the average number of spots over background and adjusted to spots per one million cells seeded. Responses are considered positive if the number of spots is: 1) at least twice greater than background, 2) greater than 50 spots forming cells per one million splenocytes over background (i.e. one response over background per 20,000 splenocytes), and 3) statistically significant by Student's t-test in comparison with the corresponding spot forming cell data set for other groups (p<0.05).

Statistics

Differences between experimental groups were compared by one-way analysis of variance with Dunnett's multiple comparison test, or the non-parametric equivalent, Kruskal-Wallis with Dunn's multiple comparison test, as appropriate (GraphPad Prism version 5.01, GraphPad, La Jolla, CA).

Results

Multi-epitope DNA vaccine construction

We designed two DNA vaccines, HelicoVax A and HelicoVax B, each of which contains a distinct set of 25 HLA class II epitopes (Figure 2 and Table 1). The epitopes were previously identified using immunoinformatics methods that selected immunogenic and conserved sequences from the H. pylori 26695 and J99 genomes [17]. Epitope sequences were randomly concatemerized, at first. To avoid production of neo-epitopes at epitope junctions, the VaccineCAD algorithm was used to re-arrange epitopes in an order that diminishes potential junctional immunogenicity. The default order for each construct contained significant predicted immunogenicity at two junctions in HelicoVax A and none in HelicoVax B (EpiMatrix scores >10). Re-ordering of epitopes by VaccineCAD and insertion of GPGPG spacers (24) produced sequences with minimized junctional immunogenicity (EpiMatrix scores < 4.5) (Supplemental Data Tables 1 and 2).

Figure 2.

Figure 2

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Outline map of H. pylori DNA vaccine construct

Table 1.

HelicoVax T-cell Epitopes. Column 1: peptide ID. Column 2: amino acid sequence. Column 3: GenBank accession number of parent protein. Column 4: sequence location in protein.

PeptideID AA SEQUENCE GenBank
Accession
Number		 Location in Protein
HP 4001 NDNVRAYGGNIYEGVLINYYKAIDYMLLNDS GI-15611087 139-173
HP 4007 RSKSKAKVRINDLRWVFSQRLSALVG GI-15611162 73-99
HP 4009 MLLERSLIRVNKERLQTYLSLYANETSTRLSEV GI-15611210 141-171
HP 4018 GLHFLGVFRFAFLYKTQSVGLASKS GI-15611320 571-595
HP 4026 MHQYKRMQTYPVLFAIQKLALENN GI-15611443 152-176
HP 4029 PAIYFANSSISGYVFLGLKTKRVRLDA GI-15611469 42-69
HP 4032 AMGFNNYGAILGVRAFNRFAPYKTPIGINLGKN GI-15611480 9-38
HP 4034 DSMIEIVNANFSLVHRYYHQKAQILGHK GI-15611489 503-529
HP 4040 YDRWHGAFRLGYTYRINPFVGIYAQ GI-15611518 165-192
HP 4042 LYFLGGVLRHARGLAAFTNASTNSYKRL GI-15611528 22-49
HP4054 SDVVRTIEAYRMLRPLVIYPF GI-15611636 340-371
HP 4055 TINVYYFNHGNLSFTYRRQYSLYVGYR GI-15611681 67-101
HP 4060 LNTWLNMNALAPKILKALYAYGAQGINLGLNL GI-15611745 177-208
HP 4061 FALVVFVLSVLLMFKQVRIWIYQYH GI-15611753 174-201
HP 4064 HFSYKRYLINTLRKEFNFLGTPLILNA GI-15611840 245-272
HP 4065 MKNFSPLYCLKKLKKRHLIALSLPLLSY GI-15611844 14-42
HP 4067 CHALYNVGRISTYMLLGAITAGLGNS GI-15611862 53-84
HP 4068 KIMYYILPQVWAWKKWRAKSLEKYCDF GI-15611868 408-438
HP 4070 QESVKDAYSEFVFALKRLKILKAQMLELQKIN GI-15611883 112-140
HP 4071 YNTYYKAGGAEVKYFRPYSVYWVYGY GI-15611916 59-85
HP 4077 REEIEFMKRKYRLMWGKVADMSSVNK GI-15612040 126-153
HP 4087 MPPIYRAENGLLVIRPLIKVREASS GI-15612173 29-58
HP 4100 CKTMALALKALGVKRAMVVNGGGTD GI-15612266 95-125
HP 4111 QKYFNDYLGLSSYGIIKYNYAQANNEKIQQL GI-15612411 31-61
HP 4117 VWPFESLPSYLQVFVQIVPAYHGISLLG GI-15612444 59-86
HP 4119 EIKVSSRQKNVALARKLVVYFARLYTPNPTLSLAQF GI-15612482 49-78
HP 4120 GSSYHASLASVYLFERLAKIRARAILASEYR GI-15612485 318-345
HP 4126 GEWYLVAMAYNYGLRKVQNAIKAAGTSD GI-15612545 82-113
HP 4127 WKKLVKRFDVNYQFIPIIKNMLIGASVPQEFL GI-15612545 47-77
HP 4152 DTAYKMLKANPTLALSAE GI-15611119 723-740
HP 4153 DMLYKLNESLRIYQNVLSNNQDQL GI-15612318 39-62
HP 4154 SNRFMLLWKNRYTLAKVQSFKLEPG GI-15612384 78-102
HP 4156 FEKKLFVMNYPLTLYTTSPYGISEE GI-15611791 56-80
HP 4157 MEQVIQNYRMIVALIQNKLSDA GI-15611555 285-306
HP 4160 PFDLKLLEKEFYQNNATSLLVTS GI-15611379 245-267
HP 4164 NMELKKALRHYLYAQTSNMVINCV GI-15611135 173-196
HP 4165 GAFYKVWKNANAYIGTTGNPLGID GI-15611410 344-367
HP 4168 INSIPGYVKLFRKAYGSKVKID GI-15612419 165-186
HP 4169 ESNISRRTAWGSWLQASAPYAIAPV GI-15611318 115-139
HP 4171 PTNFVLGANIAGFRKVASAMIAQG GI-15612066 424-447
HP 4174 LEDLLYLINRPAYANAKVSLQADF GI-15612087 159-182
HP 4175 GAIITGNYALQAKLTGALFSEDK GI-15612537 201-223
HP 4179 ELVALYQSAKALSAYWLEIE GI-15611341 413-432
HP 4181 GLIYFFLNANTPSQHGF GI-15612512 118-134
HP 4189 IKEYQYSGAFKAVFPLKVNQM GI-15612027 73-93
HP 4192 TPGLLQFYRSALAYQLRDE GI-15612041 297-315
HP 4193 FGQYKKLVNRFEGVLTGKGLTYGGS GI-15612066 176-200
HP 4197 ASPYKNMLSLTLNAANGTISVSG GI-15612049 405-427
HP 4198 LENYRSLNALFTRSLKKERPFDK GI-15612340 46-68
HP 4199 ALKIEGRTKSSYYAAQTTRIYRLA GI-15611225 250-273
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H. pylori colonization

A small number of mice died due to peri-procedural complications, so that the final numbers of mice available at study end were 18 in the lysate and DNA IM groups, 17 in the empty plasmid control group and 19 in the DNA IN group.

The results of H pylori infection density evaluated by real-time PCR are shown in Figure 3. Mice in the DNA IN group had the lowest numbers of bacteria, with 5 of the 19 mice having no detectable H. pylori DNA (compared with 2 of 17 in the pVAX group, none of 18 in the lysate group and 3 of 18 in the DNA IM group). Considerable variation in infection density within each group was evident, but the median value for the DNA IN group (1.05) was significantly lower than the median for each of the other 3 groups, lysate median 47.4, pVAX median 25, DNA IM median 53.4 (P<0.001 versus lysate, P<0.01 versus pVAX, and P<0.001 versus DNA IM).

Figure 3.

Figure 3

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H. pylori colonization. Quantification of gastric H. pylori colonization was performed by real-time PCR and expressed as the ratio of H. pylori's SSA gene to mouse GAPDH. Black bars represent median value of each group. Data analysis by Kruskal-Wallis oneway analysis of variance followed by Dunn's test demonstrate that the values for mice receiving DNA IN group were significantly lower than each of the other groups.

Histology

Histological abnormalities in these mice are shown in Table 2, including the median (range) scores for inflammation, epithelial defects, oxyntic atrophy, hyperplasia, metaplasia and dysplasia, as well as total score. Overall, the histological changes were quite mild and differences were only statistically significant for inflammation and dysplasia, when comparing mice treated with pVAX versus lysate (less advanced histological changes in the pVAX group, P<0.05 for inflammation, P<0.01 for dysplasia).

Table 2.

Gastric corpus histology was evaluated on a 0-4 scale [21] in mice euthanized 45 weeks after H. pylori-infection. Data are shown as median (range) of lesion index and analyzed by a Kruskal-Wallis one-way analysis of variance followed by Dunn's test.

Group N Median (range) of Lesion Index
Inflammation Epithelial
Defects		 Oxyntic
Atrophy		 Hyperplasia Pyloric
Metaplasia		 Dysplasia Total
Hp
Lysate 		18 1 (0.5-2) * 1 (0.5-2) 0.5 (0-2.5) 0.25 (0-2) 0.5 (0-1.5) 0.5 (0-1.5) ** 3.5(1-10.5)
pVax 17 0.5 (0-1.5)* 1 (0.5-2) 0 (0-1) 0 (0-1.5) 0 (0-2) 0 (0-1)** 1(0.5-8.5)
DNA IM 18 0.5 (0-3.5) 1 (0-2.5) 0.5 (0-1.5) 0.25 (0-3) 0.5 (0-2) 0.5 (0-2.5) 3 (0-15)
DNA IN 19 0.5 (0-1.5) 1 (0-1.5) 0 (0-1) 0 (0-1.5) 0.5 (0-1) 0 (0-1) 2 ( 0-7.5 )
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*

Significantly more inflammation (P< 0.05), and dysplasia, comparing H. pylori lysate group with pVax control.

**

Significantly more inflammation, and dysplasia (P< 0.01), comparing H. pylori lysate group with pVax control.

Immune responses

ELISpot assays of epitope-stimulated splenocytes in the DNA IN and DNA IM groups demonstrated that 33/50 epitopes (66%) stimulated >50 interferon-gamma spot forming cells per million splenocytes (Figure 4, gray bars), compared to only 2/50 epitopes (4%) which were recognized in SS1 lysate-immunized animals (Figure 4, open bars). Additionally, all peptide pools stimulated robust interferon-gamma production. There was no consistent difference between intranasal (filled circles) and intramuscular (open circles) DNA immunization on the single-epitope level, but intranasal vaccination generally was associated with stronger responses, particularly among the most immunogenic epitopes. Consistent with the colonization data, SS1 lysate and DNA IM groups, respectively, stimulated 2 and 6 statistically significant responses when compared to the pVAX group, while 23 significant responses were observed in the DNA IN group (P<0.05). pVAX alone did not stimulate any statistically significant ELISpot responses

Figure 4.

Figure 4

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Cell-mediated response to immunization of p27-deficient mice with HelicoVax A and HelicoVax B epitopes. Mice were primed with plasmid DNA vaccine and boosted with peptide pools comprising the HelicoVax A and HelicoVax B epitope sets or vaccine vehicle containing no epitopes. Splenocyte responses to individual and pooled epitopes were measured by IFN–γ ELISpot. Data are the mean spot forming cells (SFC) per million splenocytes for HelicoVax-vaccinated mice (filled bars), SS1 lysate-vaccinated mice (open bars) and HelicoVax DNA IN (filled circles) and DNA IM (open circles) groups of mice. Peptide pools 1 to 3 correspond to the epitopes contained in DNA vaccine construct A (HelicoVaxA), pools 4 to 6 to DNA vaccine construct B (HelicoVaxB).

Discussion

The results of this study suggest that a DNA-prime/peptide-boost vaccine designed to present multiple H. pylori epitopes has therapeutic efficacy when delivered intranasally to mice previously infected by H. pylori. The multi-epitope vaccine induced a broad immune response that led to a significant reduction in H. pylori colonization. In contrast, mice given a whole H. pylori lysate or an empty plasmid intranasally as the immunogen, or immunized with the same vaccine intramuscularly failed to eradicate the organism. These encouraging, though preliminary, results suggest that further development of an epitope-based mucosal vaccine against H. pylori can potentially lead to a novel approach to prevent H. pylori-associated diseases such as peptic ulcer disease and gastric cancer in humans.

Importantly, the vaccine-induced immune response parallels what is observed in natural infection. H. pylori infection dramatically influences the gastric leukocyte environment with recruitment of T cell populations, primarily the CD4+ phenotype [22], that constitute an enriched source (~1-10%) of H. pylori-specific T cells compared with peripheral T lymphocytes [23, 24]. Gastric T cells isolated from biopsies of H. pylori-infected subjects exhibit a predominantly proinflammatory Th1 phenotype characterized by a high frequency of IFN-γ-secreting cells [22, 25]. The finding that live H. pylori preferentially stimulates production of IL-12 [26], which selects for Th1 cell development, may explain the finding that the majority of Th cell clones derived from H. pylori-infected patients are comprised of Th1 effector cells [23, 27]. H. pylori pathogenesis also involves regulatory T cells (Tregs). Depletion of CD4+/CD25hi Tregs from infected human donors leads to increased H. pylori-specific memory T cell proliferation and IFN-gamma production in response to H. pylori pulsed dendritic cells. Additionally, increased levels of CD4+/CD25hi/FoxP3+ regulatory T cells have been observed in gastric and duodenal mucosa of H. pylori-infected humans [28]. Moreover, similar results have been described in mouse models of H. pylori infection [29, 30]. These findings underscore the importance of a Treg response in H. pylori immunopathogenesis and suggest that persistence of H. pylori infection may be explained by Treg-mediated suppression of pathogen-specific memory T-cells [31]. As Tregs contribute to equilibrium between host and pathogen, inflammation is maintained at low levels, allowing H. pylori to survive. Taken together, immunopathogenesis studies suggest that a vaccine that elicits a strong Th1 response, like the CD4+ T-cell epitope-based vaccine described here, should support the immune system's fight against H. pylori infection. However, we appreciate that identifying correlates of vaccine efficacy against H. pylori remains a major challenge [7].

The CDKN1B deficient mouse model was chosen for this study because it had been shown to develop gastric cancer about 1 year after H. pylori infection, following a sequence of progressive histological changes (gastritis, glandular atrophy, metaplasia and dysplasia) similar to the events that appear during gastric carcinogenesis in humans infected by H. pylori [14]. One of the aims of this study was to determine whether the therapeutic vaccine could inhibit these progressive histological changes and thus prevent cancer in this model. However histological changes in all four arms of this study were relatively mild, even in the group that received no immunogen (the pVAX group). Indeed, histological abnormalities evaluated at 45 week post-infection were, if anything, less severe in those mice that received the pVAX construct than in mice that had been exposed to H. pylori lysate or the epitope based H. pylori DNA vaccines. Although it might be assumed that reduction in H. pylori colonization will lead to less inflammation and other features of gastric damage included in the histological score, it is important to note that within groups of C57BL/6 mice successfully infected by the SS1 H. pylori strain, there is an inverse relationship between the bacterial load and the total gastritis score [32]. Another important consideration when interpreting histological changes is the phenomenon of “post immunization gastritis” in which successful elimination of H. pylori following vaccination is accompanied by a transient increase of gastritis over a few weeks, though this ultimately abates with time [33]. The current study evaluated inflammation only at the end of the experiment and therefore the kinetic changes in gastritis over the course of the 45 week infection period remains unknown; it is conceivable that post-immunization gastritis (presumably absent in the pVAX group) might account for some of the discrepancy between the histological scores and the H. pylori colonization at 45 weeks post infection.

A limitation of the current study is that we did not include a group of mice that were infected with H.pylori but were not subsequently subject to any subsequent experimental manipulations at all (a true “negative control” group). Thus, we cannot state with certainty that the DNA IM strategy or the HP lysate strategy (or indeed the pVAX control) did not reduce H.pylori colonization. We can only state with certainty that the DNA IN strategy was superior to the other 3 strategies. Indeed, because all mice in the study received LT as adjuvant, a non-specific effect of adjuvant also cannot be entirely ruled out. We suspect that the intranasal route to deliver the epitope-based DNA vaccine was more efficacious than the identical vaccine delivered intramuscularly because mucosal immunization likely produced a more robust gastric mucosal cell-mediated immune response against H.pylori. However, further studies will be necessary to directly test this hypothesis.

Despite decades of research, the immune response to H. pylori and, in particular, the correlates of successful immunity necessary for H. pylori vaccination remain poorly understood. Based upon many previous studies in animal models of infection it is known that the production of antibody in the stomach (IgA) provides little or no protection against H. pylori infection [34]. In contrast MHC Class II restricted T-cells including Th1, Th2 and Th17 cells are important in mediating immunity in murine models [7]. In humans, chronic infection by H. pylori is associated with robust immune and inflammatory responses that nevertheless rarely succeed in eradicating the organism and decades long persistence is typical, related in part to the strategy of H. pylori to survive and evade immune defenses [4].

The choice of the CDKN1B mouse as a model for testing the potential therapeutic efficacy of an epitope based vaccine against H. pylori was predicated upon increased susceptibility of these mice to gastric cancer and the opportunity to determine whether therapeutic immunization against H. pylori could reduce recurrence of this highly clinically significant endpoint. However, the deficiency of p27 in all lineages, including of the lymphoid lineages, may have limited extrapolation from this model to other murine models for H. pylori infection. For example, in contrast to wild-type mice the T cells of p27-deficient mice are more susceptible to apoptosis following interleukin-2 withdrawal demonstrating the importance of p27 in the survival of activated T-cells [35]. These mice also have reduced proliferative capacity of their T-cells, especially the CD8+ subset [36]. While p27 deficiency does not apparently affect the differentiation of T cells into Th1 versus Th2 subsets, [37] exaggerated inflammatory responses have been noted in mice populated with p27 deficient bone marrow [38] suggesting that deficiency of p27 in bone marrow-derived progenitor cells can promote inflammatory responses. Furthermore, two groups have demonstrated failure of T-cell tolerance in p27-deficient mice [39, 40].

Taken together, these results demonstrate subtle but potentially important abnormalities in the immune system of p27-deficient mice that should be borne in mind when extrapolating from the current results to those that might be expected in wild type mice. Future studies are planned using “humanized” HLA transgenic mice as a bridge to clinical vaccine development.

Supplementary Material

Supplemental Table 1

EpiMatrix scores at epitope junctions in HelicoVax A. Column 1: N-terminal peptide ID. Column 2: N-terminal peptide EpiMatrix score. Column 3: spacer sequence added to minimize potential immunogenicity between epitopes. Column 4: C-terminal peptide ID. Column 5: C-terminal peptide EpiMatrix score. Column 6: Junctional sequence EpiMatrix score.

Supplemental Table 2

EpiMatrix scores at epitope junctions in HelicoVax B. Column 1: N-terminal peptide ID. Column 2: N-terminal peptide EpiMatrix score. Column 3: spacer sequence added to minimize potential immunogenicity between epitopes. Column 4: C-terminal peptide ID. Column 5: C-terminal peptide EpiMatrix score. Column 6: Junctional sequence EpiMatrix score.

Acknowledgements

This work was supported by Public Health Service grants R43AI065036 (ASD), U19AI082642 (ASD) & R01CA111533 (SFM).

We appreciate the assistance of Emilia Mia Sordillo (St. Luke's-Roosevelt Hospital Center, New York, NY) for lysate preparation, John D. Clements (Tulane University, New Orleans, LA) for providing LT (R192G), and Jacques Pappo and Julie McMurry for helpful discussions on study design.

Footnotes

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Two of the contributing authors, Anne S. De Groot and William D. Martin are senior officers and majority shareholders at EpiVax, Inc., a privately-owned vaccine design company located in Providence, RI. Dr. De Groot is also a faculty member at the University of Rhode Island and Brown University.

Conflict of interest statement

These authors acknowledge that there is a potential conflict of interest related to their relationship with EpiVax and attest that the work contained in this research report is free of any bias that might be associated with the commercial goals of the company.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Table 1

EpiMatrix scores at epitope junctions in HelicoVax A. Column 1: N-terminal peptide ID. Column 2: N-terminal peptide EpiMatrix score. Column 3: spacer sequence added to minimize potential immunogenicity between epitopes. Column 4: C-terminal peptide ID. Column 5: C-terminal peptide EpiMatrix score. Column 6: Junctional sequence EpiMatrix score.

NIHMS268971-supplement-1.pdf (27.8KB, pdf)
Supplemental Table 2

EpiMatrix scores at epitope junctions in HelicoVax B. Column 1: N-terminal peptide ID. Column 2: N-terminal peptide EpiMatrix score. Column 3: spacer sequence added to minimize potential immunogenicity between epitopes. Column 4: C-terminal peptide ID. Column 5: C-terminal peptide EpiMatrix score. Column 6: Junctional sequence EpiMatrix score.

NIHMS268971-supplement-2.pdf (28KB, pdf)

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