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Drug Discovery Today: Disease Models Vol. 1, No. 1 2004

Editors-in-ChiefJan Tornell – AstraZeneca, SwedenDenis Noble – University of Oxford, UK

Infectious diseases

Models for bacterial infectious diseases:Helicobacter pyloriPaolo Ruggiero∗, Ali Alloueche, Rino Rappuoli, Giuseppe Del GiudiceIRIS Research Center, Chiron S.r.l., Via Fiorentina 1, I-53100 Siena, Italy

Helicobacter pylori infects >50% of the human popula-

tion, causing chronic inflammation of the gastric mu-

cosa and severe gastroduodenal diseases. As current

therapies face problems of poor compliance and in-

creasing antibiotic resistance, vaccines have been inves-

tigated. Animal models allow the mechanisms of patho-

genesis and immune responses to be studied; however,

protective efficacy found in animals has not been con-

firmed in some clinical trials, and the nature of protec-

tive immune responses is still to be clarified. In silico and

in vitro studies can help us to identify and investigate

new virulence factors and mechanisms of protection.

IntroductionHelicobacter pylori is a spiral-shaped,Gram-negative bacteriumthat chronically infects the gastric mucosa of >50% of the hu-man population, causing chronic inflammation of the stom-ach and development of gastroduodenal diseases, such as gas-tritis, peptic ulcer and gastric cancer.The prevalence of infection varies with geography and

socio-economic conditions, and it is higher in developingcountries. H. pylori infection is acquired early in life, mostlywithin the family. The risk of infection decreases duringchildhood in developed countries, probably reflecting bettersanitation and living conditions.The current antibiotic-based therapies against H. pylori are

generally effective in 80–90% of cases. The main reasons oftherapy failure are increasing antibiotic resistance and lack

∗Corresponding author: (P. Ruggiero) paolo ruggiero@chiron.it

Section Editor:

Rudi Balling—German Centre for Biotechnology,Braunschweig, Germany

The attention that Helicobacter pylori received changed dramaticallywhen this bacterium was linked to the pathogenesis of gastritis andstomach cancer. In the meantime, a great deal has been found aboutindividual virulence genes of this pathogen and some of themechanisms involved in the crosstalk between the microbe and itshost. A complete picture of the infection process is still missing,particularly insight into the determinants that lead to asymptomaticchronic infection or to progression to gastritis and even cancer insome individuals but not in others. Paolo Ruggiero and his colleaguesfrom the IRIS Research Center of Chiron in Siena are experts in thebusiness of vaccine development and therefore ideally suited toprovide us with an overview of the suitability of animal models for thepurpose of understanding the pathogenesis of H. pylori-related diseaseand its prevention through vaccine development.

of patient compliance, which can lead to discontinued treat-ment. To overcome these drawbacks, efforts are being in-vested in the development of an effective vaccine.

In vitro modelsIn vitro studies of the interactions of eukaryotic cells with ei-ther H. pylori living cells or purified toxins have contributedto the understanding of the mechanisms of the pathogen-esis, and can further help us to elucidate toxin properties,or to investigate substances that can interfere with thebacterium/toxin activities.Cultured human gastric explants have been used to study

cytokine responses to H. pylori strains or antigens, show-ing a production of growth-related oncogene (GRO)-� andpro-inflammatory cytokines [1].The ability of H. pylori to adhere to gastric epithelium has

been studied with human cell lines such as HEp-2 (larynx epi-

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Glossary

CagA: a polypeptide with molecular mass of ∼120–130 kDa encodedby cagA gene (Cytotoxin Associated Gene A), containing a variablenumber of tyrosine phosphorylation motifs.cag PAI: cag pathogenicicty island (PAI) is a ∼40-kilobase region of theH. pylori genome that encodes Type IV secretion system and CagA toxin.HP-NAP: H. pylori neutrophil activating protein, with molecular massof ∼17 kDa, encoded by napA gene. The native protein oligomerizesinto dodecamers.Type IV secretion system (T4SS): T4SSs are cell envelope-spanningcomplexes that are believed to form a pore or channel through whichDNA and/or protein translocates from the donor cell to thecytoplasm of the recipient cell. T4SS is well known in bacteria such asEscherichia coli and Agrobacterium tumefaciens. H. pylori T4SS apparatus,encoded by cag PAI, injects CagA toxin into gastric epithelial cells.Urease: the enzyme urease catalyses the hydrolysis of urea to carbondioxide and ammonia. H. pylori urease consists of two subunits, ureAand ureB, encoded by ureA and ureB genes, with molecular mass of∼30 and ∼65 kDa, respectively. The urease activity increases local pH,thus enabling H. pylori survival in the acidic gastric environment.VacA: vacuolating-associated cytotoxin A, encoded by vacA gene,expressed as a ∼140-kDa precursor and secreted as a ∼95-kDamature protein that is further cleaved into two moieties, 37 and58 kDa, that remain associated. Native VacA oligomerizes into bothhexa- and hepta-mers.

dermoid carcinoma), AGS (gastric adenocarcinoma), Caco-2(colon adenocarcinoma) and MDCK (kidney). H. pylori canenter and survivewithinmultivesicular vacuoles of these cells[2], thus constituting a reserve of living bacteria.H. pylori typeI strains, containing cag PAI, encoding a type IV secretionsystem and CagA toxin (see Glossary), are often associatedwith the most severe pathological outcome of the infectionand elicit interleukin (IL)-8 production on AGS cells. CagA istranslocated within AGS cells, where after phosphorylationit induces dramatic changes of cellular shape and orientation[3]. In addition, CagA targets H. pylori cells to host cell inter-cellular junctions and disrupts junction-mediated functionsin both AGS and MDCK cell cultures [4].The H. pylori VacA toxin (see Glossary) induces vacuola-

tion in cultured HeLa cells and damages gastric epitheliumin vivo. In a model of artificial membranes, VacA formsanion-selective channels, indicating that the vacuolating ac-tivity derives from osmotic imbalance of intracellular acidiccomponents [5]. In mouse bone marrow derived mast cells,VacA has chemotactic activity, and induces production ofpro-inflammatory cytokines [6]. Moreover, it blocks prolifer-ation of T lymphocytes by inducing a G1/S cell cycle arrest,and inhibits their activation by down-regulating IL-2 tran-scription, as shown both in human CD4+ T cells and inJurkat human T cell line [7].Monolayers of T84 human intestine cell line have allowed

studying the polymorphonuclear leukocyte (PMNL) responsetoH. pylori, showing that cag PAI, but not VacA, plays a pivotalrole in PMNL transepithelial migration [8].

Infiltration of neutrophils and mononuclear inflamma-tory cells within the H. pylori-infected stomach mucosa iscommon. In vitro studies have led to the characterization ofHP-NAP toxin (see Glossary), which induces production ofreactive oxygen radicals by neutrophils, is chemotactic forhuman neutrophils and monocytes, and activates mast cells[5].Upon stimulation with H. pylori preparations, peripheral

blood mononuclear cells (PBMC) from H. pylori-negativedonors produce Th1- rather than Th2-type cytokines [9].It has been reported that H. pylori resists phagocytosis

by macrophages [10] and/or interrupts phagosome matura-tion [11]. However, we have shown that both fresh murinemacrophages and the mouse phagocytic cell line GG2EE ex-ert a complement-independent, antibody-dependent killingactivity on H. pylori [12].

In vivo modelsAnimal models allow bacterium–host interactions, and themechanisms of immune responses to infection or vaccinationto be studied. Despite the unavailability of an ideal animalmodel reproducing all aspects of H. pylori infection and dis-ease observed in humans, the existing models, which rangefrom rodents to primates, represent valuable tools for improv-ing strategies againstH. pylori infection [13,14]. Experimentalinfection in animals is achieved by oral or intragastric admin-istration of bacteria.

MiceMost of the in vivo studies withH. pylorihave been undertakenin rodents, especially in mice. Following the first models ofeither athymic (nude) or euthymic germ-free mice, infectionwas achieved in BALB/c mice. Then, stable and reproducibleinfectionmodels in immunocompetent mice were developed[14]. Generally, theH. pylori strainsmust be adapted tomouseby repeated cycles of infection. However, suckling mice havebeen reported to support colonization of a wide range of H.pylori strains, even non-motile [15]; this model allows colo-nization defects inH. pylori strains to be identified, andmightbe useful for studying immunity to primary infection.The mouse model reproduces several aspects of human in-

fection, namely, the infection persists chronically with thedevelopment of gastritis and gastric hyperplasia [13]. Gastricpathology is much more severe in C57BL/6 than in BALB/cmice [14], suggesting an important role of host immunity.BALB/cmice also developmucosa-associated lymphoid tissue(MALT) lymphoma [13].Mouse models remain a valuable model in unravelling the

mechanisms of pathogenesis. For example, VacA has beenshown to behave as a ligand for protein tyrosine phosphatasereceptor type Z (PtprZ) inducing gastric ulcers by erroneoussignaling, as PtprZ-deficient C57BL/6 mice do not show mu-cosal damage by VacA [16]. An important demonstration of

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the role of host immune response against H. pylori in thedevelopment of gastric ulcers has been achieved using severecombined immunodeficient (SCID) CB-17 mice, which de-velop gastric ulcers upon intraperitoneal injection of periph-eral blood mononuclear cells (PBMC) from H. pylori-infectedpatients withMALT lymphoma [17]. The pivotal role of IFN-�in promoting H. pylori-induced gastritis has been proven bythe observation that infected IL-4-deficient C57BL/6 micedevelop a more severe gastritis and show higher levels ofIFN-� production by stimulated splenocytes, compared withwild-type controls, whereas IFN-� deficient mice do not de-velop gastritis and their splenocytes produce higher levelsof IL-4 [18]. The involvment of CD25+ T cells in reducingimmunopathology, possibly by reducing the activation ofIFN-�-producing CD4+ T cells, has been demonstrated inathymic C57BL/6 nu/numice reconstituted with lymph node(LN) cells or LN cells depleted of CD25+ T cells (CD25− LN).After H. pylori infection, mice transferred with CD25− LNshow higher gastritis than mice transferred with LN. Stim-ulated splenocytes from mice receiving CD25− LN producethe highest level of IFN-� [19].Several vaccination studies have been performed on mice

[13] with varying success ranging from complete eradicationto reduction of bacterial colonization to a variable extent.Recently, successful systemic vaccination againstH. pylori hasbeen reported in neonatal C57BL/6 mice [20], suggesting asimilar approach for human vaccination.H. pylori infection elicits a strong immune response; nev-

ertheless, the infection is rarely cleared. The mechanismsof protective immunity against H. pylori have not yet beendefined. In the past years, the preferential association ofpro-inflammatory Th1-type responses with H. pylori infec-tion, and of Th2-type responses with a protective responsefollowing vaccination have been proposed. More recent ob-servations in immunodeficient or knockout mouse modelsindicate that the regulatory roles of Th1 and Th2 cells inprotective immunity against H. pylori are still to be clarified.Antibody response is not required for protection, which isinstead mediated by CD4+ type 2 cells, as protective immu-nization can be achieved in B-cell deficient (�MT) C57BL/6mice, and protective immunity can be achieved in immun-odeficient C57BL/6 rag1−/− recipients that received splenicCD4+ T cells from alum-adjuvanted immunized mice [21].The role of cellular responses in clearing the infection andresolving gastritis is confirmed by the finding that H. pyloriinfection does not cause gastritis in severe combined immun-odeficient (SCID) C57BL/6 mice; upon adoptive transfer ofsplenocytes from infected C57BL/6 mice, infected SCID micefirst develop severe gastritis, then resolve it and clear theinfection [22]. The hypothesis that Th2 response is requiredto achieve protection is not supported by the finding thatprotective immunity is achievable in IL-4R� chain-deficientBALB/c mice [23]. Moreover, protection can be achieved in

IL-5-deficient C57BL/6 mice, as well as in double knockoutfor IL-4 and antibodies [24], and IL-12 deficient C57BL/6mice are more permissive to H. pylori colonization [25]. Fi-nally, IFN-�- or iNOS-, but not IL-12p40-deficient C57BL/6mice, can be protected by immunization [26]. C57BL/6knockout mice have been used to demonstrate that lackof IL-4, IL-10, IL-12, IL-18 or Toll-like receptor 2 decreasesthe susceptibility to colonization to a various extent, IL-10knockouts becoming almost completely resistant, whereasTNF-� receptor knockout mice are overcolonized [27]. Con-versely, IL-4- or IL-12-deficient BALB/c mice are more sus-ceptible to colonization. Vaccination of these mutants givesefficacy comparable to the wild-type strains, with the ex-ception of IL-10 and IL-12 knockout C57BL/6, in which thecolonization, already low, cannot be reduced further [27].It appears now that a Th1-type response can protect against

H. pylori infection, provided it is not prolonged, and that afine-tuning of protective immunity is exerted by Th2 cells.Thus, a protective immunization should produce a properlybalanced Th1/Th2 response [28].Very recently, in the mouse model of H. pylori infection,

microarray technique was successfully applied for the firsttime in an attempt to define an immunological correlate ofprotection. In this study, it has been shown that, in immu-nized BALB/c mice, protection correlates with increased tran-scription of genes encoding adipocyte-specific factors [29].This finding not only provides a valuable contribution to theunderstanding of the mechanisms of protective immunityagainst H. pylori but also suggests, more generally, that mi-croarraysmight be a new, powerful tool for establishing novelcorrelates of protection for the immune response.

Mongolian gerbilsMongolian gerbils are of special interest because upon H. py-lori infection they develop gastric atrophy, intestinal meta-plasia and eventually gastric adenocarcinoma [13]. In thismodel, 960 signature tagged mutagenesis (STM) H. pylorimu-tants have been screened, leading to the identification of bothnovel and hypothetical genes essential for gastric coloniza-tion [30].

Gnotobiotic pigletsThe gnotobiotic piglets model has been useful in demonstrat-ing that H. pylori urease (see Glossary) is a virulent factor invivo. However, upon infection the gastric pathology signifi-cantly differs from that observed in humans. Moreover, theseanimals require sophisticated housing facilities [14].

MonkeysH. pylori spontaneously infects non-human primates, causinga gastric pathology very similar to that observed in humans.Vaccinations in monkeys have been carried out with varyingsuccess [31]; however, high costs and difficulties to find an ap-

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preciable number of noninfected individuals limit its use. Im-portantly, H. pylori-infected Japanese monkeys, like humans,show mutations in p53, which are thought to be involved inthe pathway leading to dysplasia or carcinoma [32].

DogsFirst gnotobiotic, then conventional Beagle dogs have beensuccessfully infected with H. pylori, developing gastritis andclinical symptoms similar to those of infected humans [14].The conventional dog model can be complicated by the nat-ural infection by other Helicobacter species. However, in ourexperience, such an infection can delay, but not prevent theestablishment of an experimentalH. pylori infection. Vaccinesbased on purified antigens have been tested in this model inour laboratories with encouraging results (unpublished).

In silico modelsThe aim of in silico models is to complete the informationprovided by in vitro and in vivomodels, but most importantlyto identify new genetic determinants that mediate virulence,to be used as targets for vaccine development. The strengthof in silico methods lays in the generation of a large amountof data in a very short time, and exploiting the data obtainedby using the most advanced technologies in genomics andproteomics.In silico approaches have been used successfully to iden-

tify new vaccine candidates for serogroup B Neisseria menin-gitidis and Haemophilus influenzae [33]. For H. pylori, in silicomethods have predicted a large number of membrane- andsurface-associated proteins. Among these, 10 proteins havebeen selected as potential vaccine candidates, of which themajority had already been identified by conventional meth-ods in the pre-genomic era [33]. This exercise demonstratedthe feasibility of using in silico-based criteria as a powerfultool for the identification of vaccine candidates for H. pylori.The success of genomics-based strategies for vaccine develop-ment is, however, highly dependent upon the criteria usedfor the in silico selection of open reading frames (ORFs) thatencode potential candidate antigens. A better definition ofthese criteria should result in an improved prediction of pro-tein localization and function.Proteomics has also been successfully applied in H. pylori

studies. In fact, the H. pylori genome was the first to be usedfor protein–protein interactions studies [30]. Initial screen-ing using 2D SDS-PAGE andwestern blotting against patients’sera identified 14 novel proteins [33]. The advent of massspectroscopy as a novel analytical tool following preparative2D SDS-PAGE of H. pylori protein preparations has led to theidentification of 40 protein species, 15 of which were mem-brane or membrane-associated proteins [33]. Despite theseadvances, important information on the function of theseproteins remains missing.

Related articles

Boneca, I.G. et al. (2003) A revised annotation and comparativeanalysis of Helicobacter pylori genomes. Nucleic Acids Res. 31, 1704–1714

Joyce, E.A. et al. (2002) Redefining bacterial populations: apost-genomic reformation. Nat. Rev. Genet. 3, 462–473

Svennerholm, A.M. (2003) Prospects for a mucosally-administeredvaccine against Helicobacter pylori. Vaccine 21, 347–353

Montecucco, C. and Rappuoli, R. (2001) Living dangerously: howHelicobacter pylori survives in the human stomach. Nat. Rev. Mol. Cell.Biol. 2, 457–466

The use of the yeast-double hybrid system to studyprotein–protein interactions [30,33], combined with in vivoscreening of H. pylori mutant libraries could identify im-portant virulence elements that might represent vaccinetargets.

Model comparisonThe in vitro models are valuable tools that provide informa-tion on themechanisms of action of theH. pylori toxins. How-ever, these models allow investigation of the details ratherthan the general aspect of bacterium–host interactions.Conversely, in vivo models provide more general informa-

tion. In particular, the murine model is the most widely used.It has given a large amount of information on the pathogen-esis, the mechanisms of immune response, and the efficacyof vaccine candidates. In some cases, the investigation intothe mechanisms of immune response to infection or vaccina-tion has given controversial results, due not only to the pos-sible redundancy of somemechanisms, but also to fine differ-ences between theH. pylori strains and/or between themousestrains used. One limitation to the use of the mouse model isthat under normal conditions H. pylori-infected mice do notdevelop gastric cancer [13]; however, this is not the case forgerbils and monkeys, which are relevant in studying this as-pect of the disease.

Links

� Helicobacter Foundation: www.helico.com� European Helicobacter Study Group: www.helicobacter.org� Helicobacter pylori Research Laboratory (Barry Marshall):

www.hpylori.com.au� Centers for Disease Control and Prevention (CDC): Helicobacterpylori: www2.cdc.gov/ncidod/aip/HP/hp.asp

� WHO: State of the art of new vaccines: research and development:www.who.int/entity/vaccine research/documents/new vaccines/en/index8.html

� National Digestive Diseases Information Clearinghouse (NDDIC):H. pylori and peptic ulcer:www.niddk.nih.gov/health/digest/pubs/hpylori/hpylori.htm

� The Institute for Genomic Research (TIGR): Helicobacter pylori26695 genome page:www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=ghp

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Table 1. Comparison summary table

In vitro models In vivo models In silico models

Pros Elucidate bacterium–host cellinteractions, and give details onthe mechanisms of pathogenesisand toxin properties.

Reproduce several aspects of thehuman pathology.Allow the study ofbacterium–host interactions,pathogenesis and immuneresponses to infection and/orvaccination. Various facets ofthe immune response and ofthe cellular biology at the hostlevel can be dissected, thanks tothe availability of transgenicand knockout mice.

Can identify new potentialvaccine targets previouslyunidentified by conventionalmethods.

Cons Do not reproduce thecomplexity of the in vivoinfection.

An animal model thatreproduces all the aspects of thehuman infection and pathologyis not available; more than oneanimal model must be used.

Depending on the criteria used,can fail to identify somevaccine targets.

Ethical considerations.

Best use of themodel

Study of specific bacterium–hostcell interactions.Study of toxin activities.

Study of pathogenesis andimmune response to infection.Evaluation of vaccinecandidates.

Vaccine candidate prediction

How to get accessto the model

Literature LiteratureContacting the originators

Literature

Relevant patents n/a n/a n/a

References [1–12] [13–32] [30,33]

n/a, not available.

Pre- and post-genomic techniques benefit from knowl-edge of the genomes and in silico prediction. Interestingly,similar conclusions are rarely made when each of these ex-perimental techniques is applied to the whole genomes. Wecan consider each of these techniques as a narrow windowthat allows the visualization of a part of the picture contain-ing the biological information. Depending on the windowused, a different subset of the biological picture appears[30]. The rationale currently applied is a combination of allthese methods to maximize the output and the accuracy,but most importantly, the selection of vaccine targets basedon the biological understanding of the pathogen. Vaccinecandidates for H. pylori identified by in silico methods willhave to pass rigorous in vitro and, more importantly, in vivotesting. See Table 1 for pros and cons of the three types ofmodels.

Model translation to humansStudies in animals have allowed the testing of selected pro-tective antigens in humans. Animal studies, however, are notnecessarily predictive of what will happen in humans. In fact,vaccination strategies that have been successful in animalmodels can give unexpected results when applied to humans,as the mechanisms that confer protective immunity to H. py-lori have not yet been fully understood. This is the case of

some urease-based vaccines that had shown good efficacy inmice, reducing both colonization and gastric pathology, butfailed when tested in humans [14]. Thus, presently, the eval-uation of vaccine efficacy can only be undertaken in the con-text of large phase III clinical trials.

ConclusionsSince the revolutionary finding that chronic gastritis andulcer, and eventually gastric cancer, are caused by H. py-lori infection, big efforts have been invested in the fightagainst this pathogen. In vitro and in vivo studies have ledto the identification of some H. pylori antigens that arerelevant in its pathogenesis. Some of these antigens havealready been or are being tested in humans as a potentialvaccine. In spite of promising results previously obtainedin animals, those obtained to date in humans are notsatisfactory.Further in vitro and in vivo studies, possibly integrated into

in silico models, are still required to enhance the knowledgeof H. pylori pathogenesis and of the mechanisms of protec-tive immunity, and can help us to identify further vaccinecandidates and/or vaccination strategies. Also, for the futuredevelopment of vaccine it will be essential to exploit the newtechnologies and to define the novel correlates of protectionfor the immune response.

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Outstanding issues

� What are the protective immune responses?� What are the correlates of protection for the immune response?� Why vaccines that are protective in animals fail to confer protection

to humans?� Which route of vaccine administration gives better protection?� How to implement in silico models to achieve more reliable selection

of protective antigens?

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