The quest for a vaccine against Helicobacter pylori: how to move from mouse to man?

Forum in immunology

The quest for a vaccine against Helicobacter pylori:how to move from mouse to man?

Paolo Ruggiero, Samuele Peppoloni, Rino Rappuoli, Giuseppe Del Giudice *

IRIS Research Center, Chiron Srl, Via Fiorentina 1, 53100 Siena, Italy

Abstract

Several lines of evidence from experimental animal models of infection have clearly demonstrated the feasibility of a prophylactic andtherapeutic vaccine against Helicobacter pylori. However, comparatively few clinical studies have been carried out to evaluate whether thepositive results obtained in animals can be reproduced in humans. The preliminary results obtained with single component, mucosallydelivered vaccines have shown very limited results thus far. Very good immunogenicity and safety profiles are now being obtained withparenterally delivered, aluminium hydroxide-adjuvanted multicomponent candidate vaccines. For sure, better vaccine formulations, betterantigen preparation(s), better adjuvants, and better delivery systems have to be designed and tested for safety and immunogenicity. Thesestudies are also needed for deciphering those aspects of the effector immune responses that correlate with protection against H. pylori infectionand disease.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Helicobacter pylori; Immune response; Vaccines

1. Why a vaccine against Helicobacter pylori?

Vaccines represent the most cost-effective and successfulapproach to prevent infectious diseases. Mortality and mor-bidity due to several infectious diseases have been pro-foundly affected by the widespread use of vaccination.Smallpox has been eradicated for more than two decades.Paralytic poliomyelitis has disappeared from the Americas,Europe, Australia, and from several countries of other conti-nents, and represents the next target for worldwide eradica-tion. The introduction of conjugated vaccines againstHaemohilus influenzae type b (Hib) and, more recently,against Neisseria meningitidis group C has significantly re-duced the numbers of cases due to these pathogens.

However, several microbial agents that cause a huge bur-den of mortality and morbidity are still waiting for vaccines.One of these is Helicobacter pylori. Treatment of H. pyloriinfection, based on the association of one proton-pump in-hibitor and two antibiotics, has been proven to be highlyeffective in well-controlled clinical trials. Nevertheless, drugtreatment faces various orders of problems [1]. The real

efficacy of eradication treatment at the level of the generalpractitioner is not well known, and could be significantlylower that that reported in controlled studies. The high num-ber of tablets to be taken daily can seriously affect thepatient’s compliance. Side effects of these treatments are notrare. The emergence of H. pylori strains resistant to antibi-otics is seriously limiting cure rates. In some areas for someantibiotics, resistance can approach 50%. High re-infectionrates have been reported mainly from areas with high levelsof transmission. Last but not least, since treatment is onlygiven to symptomatic patients, patients without symptomswould still remain at risk of developing severe complicationsof H. pylori infection, such as atrophic gastritis and gastriccancer.

It goes without saying that these drawbacks of antibacte-rial therapy would be overcome by the use of an efficaciousvaccine, more specifically by a therapeutic vaccine which, inaddition to eradicating the infection, would provide the ap-propriate immunological memory to avoid re-infections,which drugs will never be able to induce.

Furthermore, one should not overlook the significant ad-vantages of a prophylactic vaccine able to prevent H. pyloricolonisation, thereby preventing the insurgence of pepticulcer, gastric cancer, etc. Indeed, a prophylactic vaccineagainst H. pylori has been predicted to be highly cost-effective in different studies [2–4]. In particular, the introduc-

* Corresponding author. Tel.: +39-0577-243261; fax:+39-0577-243564.

E-mail address: [email protected] (G. Del Giudice).

Microbes and Infection 5 (2003) 749–756

www.elsevier.com/locate/micinf

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.DOI: 10.1016/S1286-4579(03)00125-4

tion of a prophylactic vaccine in rich countries, like theUnited States and Japan, by the year 2010 and targeting allinfants would significantly reduce the prevalence of H. pyloriand of cases of peptic ulcer and gastric cancer [3]. It isinteresting that this vaccine would still be cost-effective if ithad 55% efficacy [2].

These studies suggest that a prophylactic vaccine againstH. pylori may not rank favourably in developing countries interms of costs and health benefits [2], unless a vaccinationprogramme of more than 10 years is undertaken [3]. Theseanalyses, however, certainly deserve additional consider-ation for two major reasons. Firstly, expensive vaccines, suchas those against hepatitis B and against Hib, are now beingsuccessfully introduced in developing countries even withinthe framework of the Expanded Programme of Immunisationof UNICEF/WHO, thanks to a new positive interaction be-tween the public and the private sectors. This may well be thecase in the near future for other potentially expensive vac-cines [5]. Secondly, the cost-effectiveness analyses done sofar have been limited to the H. pylori-attributable diseases,such as peptic ulcer and gastric cancer. They have not takeninto consideration other clinical consequences of infectionwith H. pylori, which are much more evident in developingcountries. For example, studies from Africa, Asia, and SouthAmerica have reported that early acquisition of H. pylorimay cause growth retardation [6,7] and that this infection, bycausing hypochloridria, favours an increase in diarrhoealdiseases [8], and favours enteric infections such as cholera[9] and typhoid fever [10]. It is clear that the cost-effectiveness of a prophylactic vaccine against H. pylori fordeveloping countries would increase significantly if theseclinical parameters were also taken into account.

2. What vaccine against H. pylori?

2.1. What antigens for a vaccine?

H. pylori is the first microorganism for which the genomesof two different strains have been fully sequenced. Despitethis, most of the information available so far on antigens,which could potentially be used as vaccine candidates, hasbeen obtained in the pre-genomic era [11]. Only recentlysome potential candidates have been selected through a sys-tematic proteomic approach and proven to be protective inmice [12]. There are several possible reasons as to why thegenomic approach has failed to give potential candidates forthe development of vaccines against H. pylori. The majorreason is probably the lack of immunological correlates ofprotection that may be predictive of immunity, and the lack ofeasy assays to test protection in vivo. In fact, the currentassays always require that stomachs of immunised/challenged animals undergo cumbersome microbiologicaland pathological investigations to evaluate vaccine-inducedprotection.

Nevertheless, the feasibility of prophylactic and therapeu-tic vaccination against H. pylori infection has been demon-strated by several groups worldwide in various animal mod-els, using either whole-cell preparations or selectedrecombinant antigens.

Usually, antigens are selected as potential vaccine candi-dates because they are surface exposed, thus easily attackedby the immune response; because they are abundant and,supposedly, more immunogenic; because they are well con-served in all microbial strains, thus suitable for wide protec-tion; and/or because they represent key virulence factorsimportant in the pathogenesis of the infection.

The H. pylori antigens selected as potential vaccine can-didates meet one or more of these criteria. The antigen whichhas received the most interest in the development of vaccinesagainst H. pylori has been urease. Urease is the most abun-dant H. pylori protein, representing up to 5–10% of the totalprotein content. This enzyme is involved in colonisation,since isogenic mutants lacking urease are no longer able toinfect experimental hosts, and also in gastric inflammatoryevents, since it appears to induce activation and adherence ofinflammatory cells [13]. Despite its abundance, urease doesnot appear to be highly immunogenic following naturalchronic infection with H. pylori, both at the level of specificcirculating antibodies and at the levels of CD4+ T cellsinfiltrating the gastric mucosae of infected individuals. Themost immunogenic protein of H. pylori is without any doubtCagA, a protein encoded within the cag pathogenicity island(PAI). The PAI encodes a series of proteins that assemble toform a “molecular syringe” that, upon contact of the bacteriawith eukaryotic cells, will inject CagA intracellularly [14].The tyrosine-phosphorylation of CagA and its interactionwith specific phosphatases will induce a series of cellularchanges, such as cytoskeleton remodelling, formation ofpedestals, etc., and a triggering of a series of pro-inflammatory events, such as induction of IL-8. CagA andthe cag PAI are considered important markers of severegastric pathology, based on the epidemiological observationsof association between CagA/cag PAI and severe gastricdisease, and on experimental observations in Mongolian ger-bils in which CagA-positive, but not CagA-negative strainswere able to induce severe gastric pathologies, includingcancer [15]. Several inflammatory events are triggered byanother H. pylori protein, referred to as neutrophil-associated protein (NAP). This protein, which is immuno-genic in humans, has been shown to induce chemotaxis andactivation of neutrophils and monocytes and of mast cells,and to stimulate production of reactive oxygen intermediatessynergystically with IFN-c and TNF-a. H. pylori-inducedinflammation is also mediated by a cytotoxin, called VacA,that induces cell vacuolation following binding through the58-kDa portion of the molecule. This subunit presents somesequence variability at its mid-region [16].

Other antigens have been shown to confer protection pro-phylactically and/or therapeutically in experimental modelsof infection [11], among which are antigens that share some

750 P. Ruggiero et al. / Microbes and Infection 5 (2003) 749–756

homologies with the self, such as heat shock proteins and theenzyme catalase, adhesins such as AlpA and BabB, someouter membrane proteins, and other, not better identifiedproteins.

It is not clear whether or not native conformation of theimmunising proteins is necessary to achieve protection.Studies in animals have shown that recombinant urease de-void of enzymatic activity, recombinant VacA without vacu-olating activity, and recombinant NAP unable to assemble toform dodecamers, are still able to protect mice against chal-lenge with H. pylori, suggesting that most likely, nativeconformations are not strictly required for protection. In-stead, the length of antigens could play a more importantrole. In fact, recombinant proteins of the two subunits ofVacA (p37 and p58), a short fragment A17/12 of CagA [17],known to contain immunodominant epitopes in humans, anda fusion protein fragment of 65 kDa of CagA [18] are unableto protect mice against a challenge with H. pylori, unliketheir full-length parental proteins.

For bacterial infection, a multicomponent vaccine ap-proach could be suitable in consideration of the complexityof the host-pathogen interactions. This approach would en-able the stimulation of an immune response against multipletargets. Most of the experimental work with H. pylori hasthus far been carried out with single antigen preparations.However, some improvement in protection has been reportedwhen animals were immunised with multiple antigen prepa-rations, such as heat shock proteins plus urease, VacA plusurease [11], and more recently, VacA plus CagA plus NAP.As already shown for other bacterial vaccines, such as theacellular vaccine against pertussis, it is likely that the mostpromising approach to the development of a vaccine againstH. pylori will be represented by the use of several bacterialcomponents.

2.2. What immune response to induce,and how to induce it?

As mentioned above, if extensive information is availablefor animals on the feasibility of prophylactic and therapeuticprotection against H. pylori infection, it is still not clearwhich are the effector immune mechanisms that mediate thisprotection. In other terms, it is not clear yet which arm(s) ofthe effector immune response (i.e. antibodies, CD4+ sub-populations, cytokines, etc.) should be induced by an idealH. pylori vaccine to be effective in a given target population.

H. pylori infects a quite peculiar and unique ecologicalniche, the gastric mucosa, where it remains mostly in theextracellular environment. This behaviour has led to thehypothesis that the best way to prevent or treat this infectionshould be through mucosal immunisation with the final aimof inducing effector immune responses at the mucosal level.Indeed, a large body of evidence exists in animals that mu-cosal immunisation with whole-cell vaccines or with se-lected recombinant antigens plus strong mucosal adjuvants isquite effective in preventing or treating H. pylori infection[11]. A major issue, however, is that, unlike the intestine, the

stomach does not harbour mucosal-associated lymphoid tis-sue. In contrast, the presence of organised lymphoid cells inthe gastric mucosa is a well-known consequence of H. pyloriinfection, and has even been considered as a pathognomonicsign of this infection [19]. As a consequence, a mucosalvaccine against H. pylori, and even more, a prophylacticvaccine given to individuals who have never had an immuno-logical experience with H. pylori, should be able to prime aspecific immune response at a site (e.g. intestine, or others)distant from the site (the stomach) where H. pylori-specificcells, either B or T cells, should be recruited to exert theireffector functions. This could indeed be the case at least inH. pylori-infected individuals. In fact, when subjects wereimmunised with an inactivated cholera vaccine directly at theintestinal level, vaccine-specific antibody-secreting cellswere detectable also at the level of the gastric mucosa only ifthe subjects were infected with H. pylori [20]. These dataimply that a therapeutic vaccine against H. pylori in humansmay induce the appropriate recruitment of specific cells atthe gastric level. In contrast, in the prophylactic context, anintervening post-vaccination infection with H. pylori mayprovide the right signalling for recruitment of specific Band/or T cells from the site of priming to the effector site, i.e.to the infected gastric mucosa. This could be mediated di-rectly through bacterial proteins able to induce chemotaxis(e.g. NAP and others) or through the induction of variouscytokines and/or chemokines able to attract cells at the site ofbacterial colonisation. Along this line of reasoning, the post-immunisation gastritis which has been observed by someauthors in mice protected against a challenge with H. pyloricould reflect not an immunopathological event, but simply avigorous local immune response that will eventually lead toprotection. Indeed, when these phenomena were followed upfor sufficient time, it was observed that this post-immunisation gastritis was transient and resulted in the com-plete disappearance of H. pylori from the gastric mucosa[21,22].

All this can also imply that effective protection againstH. pylori could be successfully achieved via other routes ofimmunisation, either mucosal or parenteral. In fact, the im-munisations with some antigens given via mucosal routesother than the oral route, i.e. nasal, rectal, etc., have beenshown to protect mice against challenge with H. pylori [23].A very exciting observation in the perspective of vaccinedevelopment has been that parenteral immunisation can in-deed also confer protection to animals, using various adju-vants [11], including aluminium hydroxide [11,24], i.e. thevaccine adjuvant most widely utilised for vaccines. Positiveresults of protection obtained in a dog model using themixture of three antigens, such as VacA, CagA, and NAPadjuvanted with aluminium hydroxide, led us to test thisvaccine formulation in clinical trials in human volunteers(see below). Data on protection against the mucosal infectionwith H. pylori are in agreement with those concerning othermucosal infections for which effective vaccines exist whichare given parenterally. This is the case, for instance, with

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acellular vaccines against pertussis, inactivated polio vac-cine, the conjugated-Vi vaccine against typhoid fever, etc.

Whatever the routes of immunisation to achieve protec-tion against an infectious challenge, it is commonly acceptedthat MHC class II-restricted CD4+ cells play a pivotal role inprotection, more than antibodies. This conclusion is based onexperiments in strains of mice lacking genes coding forimmunoglobulins and still able to be protected by mucosal orparenteral immunisation [25]. More controversial is the rela-tive role of Th1-type vs. Th2-type immune responses duringinfection and following vaccination. If, on the one hand,several lines of evidence during the past few years havesuggested that pro-inflammatory Th1-type responses aremore strongly associated with H. pylori infection, whereasTh2-type responses are those required for vaccine-mediatedprotection, more recent papers using IL-12 and IFN-c knock-out mice have reported that effective protection againstH. pylori may require IL-12 and an active Th1-type response[26,27].

Whatever the fine mechanisms underlying vaccine-mediated protection may be, it is difficult at present to trans-late the information obtained from experiments in animals tothe human situation. On the one hand, the use of knock-outstrains of mice may have biased the real role of each effectorarm of the immune response because these mice could havedeveloped alternative pathways to achieve immune-mediatedprotection, pathways not necessarily active, at least quantita-tively, in humans. For example, passive transfer of specificantibodies had been shown to protect mice against challengewith H. pylori [28]. In addition, antibodies are able to coop-erate with mononuclear cells in limiting the multiplication ofH. pylori in vitro, as recently shown by our laboratory [29].On the other hand, it is likely that most of the immunologicalparameters which may correlate with protection would belocalised at the site of infection, i.e. in the gastric mucosa, asite not easily accessible for routine assessment, and that theantigen-specific antibody and cellular responses detected inthe bloodstream may not necessarily reflect the status ofimmune-mediated protection.

It is, thus, important to move as soon as possible to trials inhuman volunteers to test whether the successful results ob-tained in animals can be reproduced in humans, and to learn,if possible, through which mechanisms effective prophylac-tic and/or therapeutic protection can be achieved in the natu-ral host of H. pylori.

3. What lessons were learned with the trialscarried out so far?

Compared with the large body of experience gained fromexperiments carried out in animals, relatively few clinicaltrials have been carried out so far in humans to test the safety,immunogenicity, and possible efficacy of potential candidatevaccines against H. pylori. This could be explained by theintrinsic difficulty in developing mucosal vaccines usingpurified proteins as immunogens, by the very poor immuno-

genicity of proteins after mucosal delivery, and, thus, by theneed to have optimised formulations and delivery systemsthat probably do not exist yet. In fact, with one single excep-tion (see below), all trials conducted so far have focused onthe mucosal (mainly oral) administration of these vaccines.In addition, with one exception, most of these trials withmucosally delivered vaccines have focused on the use of onesingle H. pylori antigen, i.e. the most abundant, the enzymeurease, either as a soluble protein or expressed in a bacterialvector.

3.1. Soluble recombinant urease as a vaccine

As mentioned above, purified recombinant proteins arepoor immunogens when given mucosally and require theconcomitant use of very strong mucosal adjuvants. Thestrongest of these adjuvants are bacterial toxins, such as thecholera toxin and the Escherichia coli heat-labile enterotoxin(LT), which however, induce severe diarrhoea in humans,seriously limiting their use as vaccine adjuvants. This is thereason why non-toxic mutants of these molecules are beinggenerated and tested as mucosal adjuvants [30].

The wild-type LT has been utilised in the preliminarytrials carried out in H. pylori-infected individuals using re-combinant urease, >70% of which is expressed in its multi-meric form [31]. In this study, 26 subjects received ureaseorally at doses of 20, 60, or 180 mg once weekly for 4 weeks,together with 5 µg of wild-type LT. Within 12 h after immu-nisation, two-thirds of the subjects developed diarrhoeawhich lasted for 1–2 d. Urease-specific serum IgA and circu-lating antibody-secreting cells were detectable in subjectswho received the highest doses of antigen (i.e. 60 or 180 mg).Partial reduction in the number of H. pylori colonies ingastric biopsies was observed, paradoxically, in the group ofsubjects that had received the lowest dose of urease (i.e.20 mg), thus suggesting a lack of correlation between urease-specific IgA antibodies and reduction in the bacterial load.Finally, immunisation did not induce any amelioration of thepre-existing gastric inflammation.

The reasons for the partial results of this trial on both theimmunogenicity and efficacy of the orally administered re-combinant urease are not clear. They may be linked to thevaccination regimen employed and to the liquid formulationof the vaccine, to the urease antigen used, to the low dose ofthe mucosal adjuvant employed (the diarrhoea induced byhigher doses, such as 10 µg, was too severe and forceddiscontinuation of this dosage), or to difficulties inherent inthe therapeutic vaccination itself that are not yet understood.

Following these results, the same group attempted to ame-liorate the safety profile of this vaccine by testing differentamounts of wild-type LT [32]. In a randomised, double-blind, placebo-controlled study, 42 healthy H. pylori-negative subjects were immunised orally with 60 mg ofrecombinant urease either in soluble or acid-resistant, encap-sulated form together with LT at 2.5, 0.5, or 0.1 µg. Thevaccine was given on days 1, 8, 28, and 57. In this study,diarrhoea (1–4 loose stools/day) was evident in 50% of


individuals receiving the highest dose of LT (2.5 µg), but wasabsent in those that received the lower doses of the LT. Aslightly better urease-specific antibody response was detect-able in the subjects that received the highest dose of LT.However, only one of the volunteers was found to developIgG against LT. Finally, only volunteers who received thehighest dose of LT experienced an increase in CD4+,CD45RO+, CD69+ cells, but only after a fifth oral adminis-tration of the vaccine. These data confirm, as already knownfrom in vitro assays and animal studies, that LT toxicity,immunogenicity, and adjuvanticity are dose-dependent, andthat a fine-tuning must be found in order to induce protectiveimmune responses against the vaccine without causing unac-ceptable side effects, such as diarrhoea.

Another attempt made to circumvent the safety issuesinherent in the use of the oral administration of wild-type LThas been to replace the oral route with the rectal route ofvaccination, which had been previously successfully em-ployed in mice [23]. In this study also, recombinant ureasewas used at a dose of 60 mg, administered as a rectal enemato 18 healthy, H. pylori-negative adults, together with either5 or 25 µg of wild-type LT, given three times, on days 0, 14,and 28 [33]. A strong antibody response to LT was detectablesystemically in the majority of the vaccinees, mainly in thegroup of subjects receiving the lowest dose of LT (5 µg).Only a small minority of subjects developed systemic anti-urease IgG or IgA antibody responses. However, no anti-urease or anti-LT IgA antibody was detectable in stool or insalivary samples. Finally, the urease-driven proliferative re-sponse and IFN-c production were negligible.

All these studies clearly show that oral administration ofrecombinant urease in particular, and likely of other antigens,requires the use of strong and safe mucosal adjuvants to begiven at doses sufficiently high to exert their adjuvant effects,but still unable to induce unwanted effects, such as diarrhoea.Both the oral and the rectal routes require the development ofappropriate formulations able to induce adequate protectiveimmune responses to H. pylori.

3.2. Salmonella-vectored urease

If the attempts made to develop an H. pylori vaccine basedon the use of recombinant urease have thus far given poorresults, the attempts to develop vaccines based on ureaseexpressed in Salmonella strains have given even poorer im-munogenicity results, despite the fact that good immunoge-nicity and efficacy had been previously observed with thesebacterial constructs in mice.

In one study, one or two oral administrations of 1010 CFUor more of a Salmonella enterica serovar Typhi strain attenu-ated by deletion of the phoP/phoQ virulence regulon andexpressing both subunits of H. pylori urease induced serumantibody responses to salmonella antigens (such as flagellaand LPS), but was totally ineffective in inducing any detect-able immune response to urease, even after a booster dose ofrecombinant urease plus wild-type LT [34]. Slightly better

results were reported in a subsequent study, in which sixvolunteers were immunised orally (5–8 × 107 CFU) withS. enterica serovar Typhimurium harbouring the samephoP/phoQ deletion [35]. Only one of the six volunteers haddetectable urease-specific IgA antibody-secreting cells; twoothers had slight amounts of urease-specific antibodies pro-duced in vitro by cultured peripheral blood mononuclearcells; and two subjects had some specific serum IgAantibodies detectable by ELISA but not by Western blotting.

No better results were obtained in another study carriedout in volunteers immunised orally with the vaccine strainTy21a against S. enterica serovar Typhi expressing bothsubunits of H. pylori urease. In this case also, none of thenine volunteers vaccinated with this construct developed anydetectable antibody response against urease, despite the threedoses of >109 CFU received on days 0, 2, and 4 [36].

It is clear that the bacterial vectors for H. pylori antigensstill require quite a lot of work of optimisation to enhance thedegree of intestinal colonisation by the attenuated salmonel-lae, to enhance the stability of the plasmid expressing theforeign gene(s), and to ameliorate the safety issues not re-solved yet by these constructs.

3.3. Inactivated whole-cell vaccines

The use of whole-cell vaccines against H. pylori inacti-vated via various means (sonication, formalin treatment, etc.)has been successfully demonstrated in a large number ofstudies carried out in animals immunised either mucosally orparenterally [11]. Whole-cell vaccines offer the advantage ofeliciting immune responses against a wide variety of anti-gens, although they offer the major disadvantage of contain-ing potentially dangerous components of the bacterium, suchas those, like LPS, sharing homologies with the self antigens,and able thus to induce immune responses cross-reactingwith epitopes of the host [37].

A formalin-inactivated whole-cell H. pylori vaccine wasevaluated in a phase I trial in both H. pylori-negative andH. pylori-positive subjects [38]. The vaccine, containingvarious amounts of bacterial cells, was given orally threetimes, on days 0, 14, and 28, together with 25 µg of theLTR192G mutant, which contains a glycine instead of anarginine residue in the A1 subunit of the LT, which is sup-posed to reduce its sensitivity to trypsin treatment. The firstpart of the trial was an open-label, dose-response study inwhich H. pylori-infected or -uninfected individuals receivedfrom 2.5 × 106 to 2.5 × 1010 bacterial cells together with theLT mutant. Vaccination elicited H. pylori-specific antibodyresponses only in subjects receiving the highest dose of thevaccine. The increase in IgA and IgG titres was marginal andobserved only in H. pylori-infected patients. The number ofantibody-secreting cells induced by the vaccine remainednegligible. However, some detectable responses were ob-served at duodenal level in H. pylori-negative subjects [39].The significance of this finding remains, however, unclear. Ifsome antibody response was induced only in the H. pylori-


infected patients, proliferative responses of peripheral bloodmononuclear cells and production of IFN-c were observedonly in uninfected volunteers (5 and 7 volunteers out of 10,respectively) who had received the highest dose of the vac-cine, following in vitro re-stimulation with a bacterial soni-cate (and not with a purified antigen, such as catalase). Thesecond part of the trial was a randomised, double-blind studyin which the H. pylori-infected individuals received either2.5 × 1010 bacteria plus 25 µg of LTR192G, or placebo plus25 µg of LTR192G. Vaccinated subjects had significantlyhigher IgA antibody titres in the stools than subjects receiv-ing the placebo. However, the coadministration of the vac-cine with the adjuvant only marginally affected the serumantigen-specific IgA antibody response. Vaccination ofH. pylori-infected patients did not achieve bacterial eradica-tion, since in both parts of the trial [13C]urea breath testremained positive 2, 6, and 7.5 months after vaccination [38].It is not known, however, whether vaccination affected thedegree of H. pylori colonisation of the stomach, since nomicrobiological data were reported from this study. Finally,diarrhoea occurred in five subjects out of 18 (28%) vacci-nated with the highest dose of bacteria plus the LTR192Gmutant, and in one out of three (33%) who received theLTR192G mutant. It is very unlikely that diarrhoea was dueto residual VacA activity present in the vaccine preparation,since VacA activity is very sensitive to formaldehyde treat-ment. Instead, it is very likely that diarrhoea was due to theLT mutant, which retains most of its toxic activities in vitroand in vivo in animals [30], and as reported in other clinicalstudies, for example, with a whole-cell Campylobacter jejunivaccine prepared in the same manner as the whole-cell H.pylori vaccine [40].

3.4. Parenteral, multicomponent vaccines

As mentioned above, it is well known that parenteralvaccination can confer protective immunity against mucosalinfection. This has also been demonstrated by several groups,including ours, in the case of H. pylori in experimentalinfections in various animal models. In most cases, however,the experimental vaccine consisted of an uncharacterisedbacterial lysate, instead of well-defined recombinant anti-gens. Previous experience with the acellular pertussis vac-cine had shown that effective immunity against pertussis wasachieved by the combination of different antigens participat-ing in different aspects of the pathogenesis of the infection.Based on this, we decided to pursue the development of avaccine against H. pylori consisting of various proteins, allinvolved in the virulence of the bacterium. Previous work inmice had shown that immunisation with VacA, CagA, orNAP protected animals against challenge with H. pylori[11,17]. We then tested the effect of immunisation with theassociation of these three antigens in an experimental beagledog model of infection with H. pylori, which reproducesvarious aspects of the infection in humans [41]. We foundthat the best prophylactic protection against H. pylori wasobtained after parenteral immunisation with the three anti-

gens formulated with aluminium hydroxide, the adjuvantmost widely utilised for human use (in preparation).

These observations prompted us to evaluate the safety andthe immunogenicity of an aluminium hydroxide-adjuvanted,multicomponent vaccine in human volunteers. H. pylori-uninfected individuals were immunised intramuscularlythree times, following three different immunisation regimes,with a vaccine consisting of either 10 or 25 µg each of CagA,VacA, and NAP, plus aluminium hydroxide [42]. This vac-cine was extremely safe, exhibiting only some mild andtransient effects at the site of injection not different in aspectand degree from those induced by any aluminium hydroxide-adjuvanted vaccine. The vaccine was highly immunogenic,inducing antibody responses to the three antigens in almostall individuals. Months after the last immunisation, most ofthe subjects had still-detectable antibody responses to each ofthe three antigens. Interestingly, vaccination induced verystrong and sustained vaccine antigen-driven cellular prolif-erative responses and IFN-c production [42]. Several lines ofevidence suggest that parenteral vaccination with VacA,CagA, and NAP induces strong and long-lasting immuno-logical memory. Preliminary data show that this vaccine isequally safe and highly immunogenic in H. pylori-infectedsubjects also (in preparation).

4. Conclusions and perspectives

Several studies in various experimental models of infec-tion have clearly demonstrated the feasibility of a prophylac-tic and therapeutic vaccine against H. pylori. However, com-paratively few clinical investigations have been carried out toevaluate whether the positive results obtained in animals canbe reproduced in humans. The preliminary results with mu-cosally delivered vaccines have shown that a clear answer isstill awaited. For sure, better vaccine formulations, betterantigen preparation(s), better adjuvants, and better deliverysystems have to be designed and tested for safety and immu-nogenicity. These studies should also be aimed at decipher-ing those aspects of the effector immune responses thatcorrelate with protection against H. pylori infection anddisease. On the other hand, an answer as to the feasibility ofan approach to vaccine based on parenteral immunisation canpave the way to a faster track for vaccine development. Thevery good safety and immunogenicity profiles of our multi-component vaccine, given intramuscularly, based on threekey components of the pathogenetic events of H. pyloriinfection, prove that this approach is indeed feasible. Theefficacy of the parenteral approach to vaccination againstH. pylori has to be shown now in humans, ideally in areaswith high incidence rates starting from the early periods oflife, when most of the H. pylori transmission takes place. Thefinal hope is that these vaccines will permit the elimination ofthis pathogen (and of the severe gastric diseases it induces,including gastric cancer), which has cohabited with humansfor more than 100,000 years [43].


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The quest for a vaccine against Helicobacter pylori: how to move from mouse to man?