Vaccine 24S2 (2006) S2/13–S2/19

How bacteria and their products provide clues tovaccine and adjuvant development

Gordon Dougan ∗, Carlos HormaecheThe Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK

Available online 17 February 2005

Abstract

Evidence has emerged that both vertebrates and invertebrates share innate immune pathways involved in the recognition of and the responseto micro-organisms, including bacteria and their products. As a consequence, particular degenerate products of bacteria can stimulate andmodulate immune responses and influence acquired immunity and, potentially, protection against disease. New knowledge in this field isbeginning to explain how vaccine adjuvants work and will facilitate the future development of novel adjuvants and vaccines.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Adjuvant; PAMP; TLR pathway; Lipopolysaccharide; CpG

1

giaboflmassmpbsnppsi

0d

. Introduction

The immune systems present in different eukaryotic or-anisms have been moulded by the frequent and constantnteractions that occur throughout life with micro-organismsnd their products. Simple host defence mechanisms haveeen identified in primitive life forms with the appearancef a rudimentary innate immune system already apparent inies [1–6]. The acquired, antigen-specific immune systemakes an appearance much later in evolution as multi-cellular

nimals developed complicated life-styles with longer life-pans. As the immune system became more sophisticated,elective pressure ensured the evolution of a range of im-une evasion mechanisms, present in both commensal and

athogenic microbial species, in an ongoing battle of witsetween host and potential pathogen. Although the immuneystem is an extremely complicated entity, which shows sig-ificant differences between animal species, there are exam-les of common mechanisms and effector pathways that areresent in both vertebrate and invertebrate species, and toome extent in plants. This is particularly true for the older

be recognised functionally and bioinformatically in genomesas diverse as man and fly [7].

One role of the immune system is to recognise the threator danger associated with infection. A second is to mountan amplified but controlled response to deal with the threat.Animals can show both short term non-specific immunity aswell as longer term acquired immunity to specific pathogen-associated antigens. Any innate mechanism that is designedto eliminate or kill hostile living cells or organisms has thepotential to inflict self harm. Thus, it is not surprising that theimmune system is activated specifically by the appearance ofthreat and that it can discriminate to some extent as to thenature of the threat. Hence, mechanisms must have evolvedto identify antigens or signals more likely to be associatedwith danger or infection.

Immunologists recognised early on the presence ofantigen-specific recognition pathways, both humoral and cel-lular, but they were slower to appreciate the importance of‘antigen recognition’ in more non-specific responses. Wenow know that mammalian cells can harbour multiple mech-anisms for recognising distinct molecular patterns rather

nnate immune system where conserved immune genes can

∗ Corresponding author. Tel.: +44 122 349 5381; fax: +44 123 349 4919.E-mail address: gd1@sanger.ac.uk (G. Dougan).

than specific antigens associated with potentially damagingmicro-organisms. We now refer to these as pathogen asso-ciated molecular patterns (PAMPs) [8–11]. These are usu-ally degenerate molecules such as DNA, lipopolysaccharide(LPS), RNA or flagella that are essential for many forms of

264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2005.01.104

S2/14 G. Dougan, C. Hormaeche / Vaccine 24S2 (2006) S2/13–S2/19

microbial life and are required for colonisation or infectionof the host. Perhaps we failed to identify these mechanismsearlier because of our fascination with the acquired immunesystem. This concept is supported by the fact that the exis-tence of PAMP recognition systems was first identified in fliesthat lack the acquired immune system. Whatever the reasons,we have now been able to begin to dissect these recognitionsystems and for the first time we are beginning to explainnon-specific antigen recognition and innate immunity at themolecular level.

One of the consequences of these advances is that we arealso beginning to understand much more about many earlierempirical observations on immunity and immune responses.Excellent examples of this can be found in the field of vacci-nology. The very earliest vaccines, including Jenner’s small-pox vaccine, were based on live attenuated micro-organisms[12]. The rationale of live vaccination is to mimic naturalinfection as closely as possible without inducing significantclinical complications or disease [13]. Jenner used a poxvirusderived from cattle that is now known as Vaccinia virus. In-oculation of humans with live Vaccinia, even with a singledose, is an effective method of inducing immunity to small-pox. Consequently, live Vaccinia is significantly immuno-genic.

The early successes with Vaccinia were not easy to repli-cate for other diseases. Attempts to make more live vaccineswovlrvwatbpfomc

ucwdppmdctgrsu

tempts to adjuvant vaccines employed the use of crude oils orother materials that had the effect of depositing material at thesite of injection. Unfortunately these often caused severe lo-cal irritation and pathology. More refined oil-based adjuvantsfound use as veterinary vaccines but they have only recentlybeen used extensively in humans in extended clinical studies[15].

Early investigators were quick to recognise that certainmicrobial products were able to act as adjuvants. Cell wallextracts of Mycobacterium were found to be particularly ef-fective. Whole bacteria such as Corynebacterium parvumwere also utilised in clinical studies. Attempts to refine ad-juvants based on bacterial extracts were compromised by thegeneral toxicity of the material, the inability to define theadjuvant at the biochemical, or later, the molecular level,and the lack of knowledge of a mechanism of how adju-vants worked. Advances in molecular and biochemical sci-ences allowed several significant breakthroughs to be made.Analysis of the cell wall of Mycobacterium defined mu-ramyl dipeptide as an active component [16]. Also attemptsto biochemically detoxify LPS led to the development of ad-juvants such as monophosphoryl lipid A [17]. In spite ofthese advances even today almost no adjuvants, other thanalum salts, are generally accepted as adjuvants for use inhumans.

2

tltpipgtaIti[

DtrdirtTmhpf

ere often complicated by the threat of reversion to partialr full virulence frequently associated with the use of suchaccines. This forced serious consideration of the use of non-iving vaccines based on antigens derived from microbes. Ineality in the early days this meant the use of whole, inacti-ated micro-organisms. The main difficulties of this approachere associated with either (a) lack of efficacy or protection

gainst disease or (b) unacceptable reactogenicity to vaccina-ion. The reactogenicity associated with the use of vaccinesased on non-living, whole micro-organisms frequently ap-eared as local tender swelling, associated with often severeever-like symptoms. Reactogenicity forced the developmentf purer vaccines that eliminated the unwanted reactogenicaterial but retained the so-called protective antigens asso-

iated with effective vaccination.Protective antigens, in the case of bacterial diseases, were

sually protein or carbohydrate in nature. The reactogenicomponents were materials associated with the bacterial cellalls including peptidoglycan or LPS. One unexpected andetrimental consequence of the use of purified vaccine com-onents was a loss in immunogenicity to the extent that purerotein or carbohydrate-based vaccines were so poorly im-unogenic that the level of efficacy of the vaccine was re-

uced to an unacceptable level. Ironically, at this point vac-ine developers had to consider adding back materials, givenhe name of adjuvants, which could enhance the immuno-enicity of purified vaccines without significantly increasingeactogenicity. Many materials with adjuvant activity wereubsequently identified but almost all of these failed to find ase in humans because they were reactogenic [14]. Early at-

. PAMPs and the identification of their receptors

Studies on the innate immune system led to the identifica-ion of the first PAMP receptor molecules. Proteins such asipopolysaccharide binding protein and CD14 were showno play a role in LPS binding and recognition and suchroteins were eventually shown to be involved in deliver-ng LPS-like molecules to monocytes and innate immuneathways. LPS was identified at an early stage as a pyro-en and a key activator of the fever response and the cy-okine cascade that leads to the production of proteins suchs tumour necrosis factor and mediators such as nitric oxide.ronically, studies on DNA vaccines led to the recognitionhat bacterially derived DNA had inherent adjuvant activ-ty associated with unmodified (non-methylated) CpG motifs18].

The concept that such a degenerate molecule as bacterialNA could act as a general adjuvant prompted further work

o identify the receptors that mediated such activity. This andelated efforts were greatly facilitated by the availability ofefined stimulators of the system. Parallel work on the innatemmune system of the fly led to the identification of particulareceptors that were involved in activating the pathway andhe receptors were eventually shown to bind certain PAMP’s.he fact that some of these receptors were conserved betweenammals and flies stimulated comparative analysis and we

ave now reached a stage where a family of PAMP bindingroteins, known collectively as the Toll-like receptor (TLR)amily have been identified [1].

G. Dougan, C. Hormaeche / Vaccine 24S2 (2006) S2/13–S2/19 S2/15

Certain TLR’s bind particular microbial ligand families,and the distribution of these receptors varies between particu-lar cell types. TLR4 is associated with the recognition of LPS,TLR5 with flagella and TLR9 with CpG [6]. We can expectmore members of this and related families and their ligands tobe identified. Other sets of PAMP receptors have been iden-tified that mediate recognition of PAMP’s associated withintracellular bacteria ([9,20]. Some of these bind peptidogly-can and can even differ in their ability to bind Gram-positiveas against Gram-negative peptidoglycan [21]. Now that wehave identified PAMP receptors, the associated signal path-ways and their mechanisms of action, we may be able to fur-ther characterise the innate immune system and eventuallyrationalise how adjuvants work. Once this has been achievedwe could be able to design better adjuvants with lower toxi-city. For example, it is now possible to set up small moleculescreens for compounds with receptor stimulating/inhibitingactivity and to assess the potential of such molecules as ad-juvants.

3. Bacteria as a route towards vaccine and adjuvantdevelopment

We can now see how bacteria and their products can beutilised as a route towards the development of both vaccinesatttlhwgttqm

4

voeosttemotta

genicity and reactogenicity frequently emerges. Reactogenic-ity is almost impossible to predict using in vitro cell-basedor in vivo non-clinical models so this facet can only trulybe evaluated in the clinic. A good illustration of this prob-lem is provided by attempts to develop Shigella vaccines[27–29]. Shigella is a cause of bacterial dysentery involv-ing invasion of the large intestine in humans. Shigella spp.are normally human adapted compromising work outside ofthe clinic. Since Shigella inhabits the lumen of the intestineand only invades the surface-associated cells of the intes-tine most vaccine developers have utilised live oral vaccinesin an attempt to stimulate local gut immunity and IgA pro-duction. Multiple independent attempts have been made overmany years to design a suitable live oral Shigella vaccine.Such attempts have utilised different combinations of atten-uating mutations and different strain backgrounds but to dateno live oral Shigella vaccine has been licensed, largely dueto problems associated with reactogenicity. This now mightbe explained in part by the discovery of the so-called NODreceptors that recognise the presence of intracellular pepti-doglycan [19–21].

Attempts have been made to utilise rational attenuation asan approach to generating other live bacterial vaccines againstdiseases such as typhoid and cholera [24,30–35]. Work on ty-phoid has focused on the rational attenuation of Salmonellaenterica serovar Typhi (S. typhi), the cause of human ty-ppaAeSiervZatTsatTSditbsssdgec

nd adjuvants. Bacterial vaccines can be based upon live at-enuated strains or inactivated products. Live vaccines havehe advantage that they harbour inherent adjuvanticity andhey can follow a more natural route into the body to stimu-ate immunity, potentially both locally and systemically. Theyave the disadvantage that live micro-organisms are viewedith great suspicion by clinicians, vaccine regulators and theeneral public alike, even in the era of recombinant DNAechnology and genetic engineering. Non-living vaccines inhe modern era need good molecular definition to facilitateuality control and the use of safe adjuvants to enhance orodulate immunogenicity.

. Live bacterial vaccines in the modern era

Perhaps the main reason why live vaccines were not de-eloped more frequently in the past was the perceived andften present danger of reversion to virulence. In the mod-rn era this problem has been tackled by using rationalr precise genetic approaches to the creation of attenuatedtrains suitable for use as live vaccines [13,22–26]. Muta-ions can be introduced into genes required for survival inhe host to disable the micro-organisms ability to cause dis-ase while preserving the ability to induce a protective im-une response. As we learn more about the molecular basis

f infection we have a larger selection of genes that can beargeted for inactivation (leading to attenuation). Althoughhis approach is theoretically simple it is in fact difficult tochieve in practice. The old problem of balancing immuno-

hoid [33,34,36]. Like Shigella, S. typhi is a human-restrictedathogen and the more promiscuous S. typhimurium is usu-lly used as a surrogate model for preclinical development.

prototype oral typhoid vaccine is available based on anmpirically attenuated S. typhi, known as Ty21a. Although. typhi Ty21a is efficacious, several doses are required tonduce moderate levels of protection in the field [31]. Sev-ral different candidate live oral typhoid vaccines based onationally attenuated S. typhi have entered early clinical de-elopment [32–34]. Perhaps the most advanced is S. typhiH9 that harbours mutations in aroC and ssaV [34]. aroC iscomponent gene of the shikimate pathway associated with

he endogenous production of the aromatic ring by bacteria.he availability of critically important aromatic compoundsuch as folate is tightly regulated in mammalian tissues soro-negative S. typhi are starved and grow more slowly inhe mammalian host [30]. ssaV encodes a component of aype III secretion system that contributes to the survival of. typhi within human cells, including monocytes and den-ritic cells [35]. S. typhi ZH9 has successfully been testedn Phases I and II clinical studies where it has been showno be immunogenic and well tolerated. S. typhi ZH9 coulde entering a Phase III vaccine trial in the next few years.saV is an attractive attenuating mutation for several rea-ons. ssaV mutant S. typhi are defective in their ability tourvive inside macrophages and, consequently, they are notetected in the bloodstream of volunteers following oral in-estion [34]. Further, S. typhimurium ssaV derivatives stillxhibit significant attenuation in some severely immuno-ompromised mice, suggesting they may have some value

S2/16 G. Dougan, C. Hormaeche / Vaccine 24S2 (2006) S2/13–S2/19

in the immunisation of humans with immunosuppression[25].

5. Live bacterial vaccines: adjuvanticity combinedwith delivery

As recombinant DNA technology developed throughoutthe 1970s and 1980s groups involved in live vaccine devel-opment began to realise that they could utilise this approachto create recombinant bacteria that could be used as anti-gen delivery systems. Basically the concept involved cloningand expressing a gene encoding a protective antigen from onespecies into an attenuated micro-organism that could then de-liver the heterologous antigen to the mammalian immune sys-tem [13,22,26,36,37]. The approach had the other potentialadvantage that it may be possible to simultaneously vaccinateagainst more than one disease using routes of immunisationthat can stimulate mucosal as well as systemic immunity.Early efforts in this area focused on viruses, such as Vac-cinia and adenoviruses and bacteria including enterics andMycobacteria (BCG). This concept quickly developed fur-ther to incorporate the potential delivery of mammalian im-munomodulators such as cytokines and tumour antigens (asa basis for the development of cancer vaccines) [38–40]. In-terestingly, live oral vaccines based on attenuated Salmonellaaitkotta

adpwnocWgapwdopocfcht

6. Bacterial vaccine components and adjuvantdevelopment

We have already discussed the contribution of the analy-sis of bacteria and their products of adjuvant development.Prior to the discovery of PAMP receptors there were manyreports in the literature of particular vaccine components hav-ing adjuvant activities. Indeed sub-cellular organelles suchas ribosomes and peptidoglycan derivatives have been pro-posed for many years as viable vaccines because of theirimmunogenicity. However, they are unlikely to reach com-mercial use because of their complexity and poor biochem-ical definition. Several TLR agonists are being developedfor use as adjuvants and as stand-alone immunomodula-tors because of their ability to stimulate both the innate andadaptive immune responses. Perhaps the derivative that hasprogressed the furthest clinically is monophosphoryl lipidA, a chemically modified derivative of Salmonella Min-nosota LPS that retains some immunostimulatory activity butwhich has significantly reduced toxicity compared to natu-ral LPS [17]. Monophosphoryl lipid A can be used aloneor in combination with other adjuvants in vaccine formu-lations [53]. This compound has been steered through pre-clinical and into clinical studies. Monophosphoryl lipid Ahas proven to be safe and effective as a vaccine adjuvantin over 120,000 human doses. It has also been used as anatpmapSa

7

bmtdcagscscftcfls

nd Shigella have been shown to be able to deliver plasmidsncorporating antigens expressed from eukaryotic promoterso the mammalian immune system [41–44]. Essentially eu-aryotic expression plasmids are introduced into Salmonellar Shigella vaccine strains and these constructs can be usedo orally vaccinate mice. This approach also offers some po-ential as a gene delivery system for non-vaccine related ther-pies.

S. typhi strains, such as ZH9, Ty21a and CVD908-htrAnd surrogate S. typhimurium equivalents have been used toeliver a variety of different antigens derived from differentathogens [45–47]. Early efforts to obtain optimal deliveryere compromised by instability associated with recombi-ant plasmids used as expression platforms for the heterol-gous antigens so plasmid stabilisation, selection and evenhromosomal integration systems were developed [48–50].ork on optimising the expression of the heterologous anti-

en focused on the utilisation of bacterial promoters that werectivated once the vaccine strain entered host tissues. This ap-roach offered the advantage that foreign gene expressionas minimised during vaccine production but maximiseduring immunisation. Promoters, such as ssaG (a componentf the SPI-2 Type III secretion system of Salmonella), werearticularly attractive because they were optimally activatednce the bacteria had entered immune cells such as dendriticells or macrophages [35,51,52]. Vaccine development ef-orts utilising S. typhi as a carrier have proceeded into thelinic [45,46], although to date mainly non-optimised strainsave been used. Further work will be needed in the clinic ifhis area is to ever reach commercial fruition.

djuvant with anti-allergy vaccines. A further class of syn-hetic lipid A mimetics, the aminoalkyl glucosaminide 4-hosphates, have been created that specifically to target hu-an TLR4 and are showing promise as vaccine adjuvants

nd as therapeutic agents capable of eliciting non-specificrotection against a wide range of infectious pathogens [54].uch compounds may herald a new generation of engineereddjuvants.

. Mucosal adjuvants and bacterial toxins

As we have discussed above many molecules that har-our adjuvant properties can bind directly or indirectly toammalian cells via specific receptor interactions. Thus, cell

argeting can enhance this activity. Adjuvants have been tra-itionally used to enhance the immunogenicity of vaccineomponents delivered by injection into the skin, i.e. parenter-lly. Soluble antigens are also frequently poorly immuno-enic when delivered to the mammalian host via mucosalurfaces. Indeed, the mucosal delivery of soluble antigensan also be used to actually set up a state of ‘tolerance’ to theame antigen delivered parenterally [55]. The current con-ept is that mucosally delivered antigens can stimulate de-ault non-responsive immunity in terms of antibody produc-ion. Of course, the mucosal delivery of vaccines is furtheromplicated by the fact that a dilution effect is encounteredollowing mucosal immunization as the antigen encountersuminal body fluids, which also harbour denaturing agentsuch as acids and proteases.

G. Dougan, C. Hormaeche / Vaccine 24S2 (2006) S2/13–S2/19 S2/17

Interestingly, certain soluble antigens have been identifiedthat not only are immunogenic when delivered via mucosalsurfaces, but also appear to act as mucosal adjuvants. Perhapsthe classic examples of this type of molecule are the bacterialenterotoxins cholera toxin (CT, produced by Vibrio cholerae)and heat-labile toxin (LT, produced by enterotoxigenic Es-cherichia coli) [55]. The simple mixing of small amounts ofLT or CT with mucosally administered antigens can greatlyenhance their immunogenicity in terms of their ability to stim-ulate the production of local IgA and systemic IgA and IgG[56,57]. LT and CT act as mucosal adjuvants in several ani-mal species, although their high toxicity for some mammalsprecludes their clinical use, particularly in humans [58]. LTand CT can be effective adjuvants following delivery of anti-gen through different mucosal routes including intranasal,intragastric or intravaginal. The molecular mechanisms bywhich these toxins work as adjuvants are not fully under-stood and may be multiple. What they appear to do is alterthe immunological environment in which mucosally admin-istered adjuvants are recognized and processed. They mayalter the behaviour of mucosally associated regulatory cellsand/or antigen presenting cells that naturally work throughthe default pathway of non-responsiveness unless a ‘danger’signal tells them to do otherwise.

The clinical development of LT and CT as mucosal ad-juvants has been compromised by their toxicity, particularlyidAmttwomoa[wchcdgttohtd

8

a

and it is highly likely that other adjuvant molecules willbe identified in the future. The challenge in the field willbe to design molecules that preferentially activate discreteimmune pathways in vivo in out bred populations. The ad-vantage of such approaches is that toxicity or reactogenicitycould be avoided. Many research teams and companies arenow using molecular screening programmes to identify po-tential lead molecules that fit this description using variousreceptors, including TLR’s in their assays. Molecules suchas LPS derivatives can serve as model molecules for such in-vestigations. Attempts to use genetic engineering to improvevaccines have also been initiated. We mentioned above thatthere are some concerns about the use of CT and LT as ad-juvants because of their toxicity and their potential to targetneurons. Lycke and colleagues have taken the innovative ap-proach of re-engineering CT by replacing the B subunit of thetoxin with the DD peptide repeats of protein A from Staphylo-coocus aureus. Consequently the toxin targets antibodies andsubsequently Fc receptors on B cells, not GM1-gangliosides[64]. The recombinant molecule, known as CTA1-DD, stillharbours the ADP-ribosylation activity of CT, has no or un-detectable levels of toxicity but still retains adjuvant activity,particularly if formulated with ISCOMs [65,66]. CTA1-DDis being prepared for a series of clinical trials and may rep-resent the prototype of a new generation of vaccines suitablefor both mucosal and parenteral use.

A

R

[

n humans were as little as few micrograms of toxin can in-uce severe diarrhoea after oral ingestion by volunteers [59].ttempts have been made to separate the toxicity of theseolecules from their adjuvanticity using site directed mu-

agenesis of the genes encoding the toxins. Several mutantoxins have been identified that have reduced toxicity buthich retain significant adjuvanticity [55,60–63]. Examplesf such toxins include LTK63, which harbours an inactivatingutation associated with the ADP-ribosyltransferase activity

f the protein. LTK63 folds into the multimeric LT moleculend is structurally almost identical to the wild-type LT toxin55]. Hence, immunogenicity and structure are retained asell as the cell targeting activity of the molecule. Further

omparisons between different classes of LT and CT mutantsave suggested that both the LT-A and the LT-B subunitsontribute to adjuvanticity [61]. Although ADP-ribosylationoes not appear to be essential for adjuvant activity it canreatly enhance it, possibly through some signal amplifica-ion process. LTK63 is being assessed in the clinic, but fur-her issues have been raised about the potential human usef these toxin-derivatives as they can target gangliosides onuman nerve cells, and in the nose they have been reported toravel up olfactory nerves into the brain, potentially causingangerous inflammatory responses [58].

. Future considerations

It is clear that bacteria and their products have providedrich source of materials with potential adjuvant activity

cknowledgement

This work was supported by The Wellcome Trust.

eferences

[1] Aderem A, Ulevitch RJ. Toll-like receptors in the induction of theinnate immune response. Nature 2000;406:782–7.

[2] Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Im-munol 2001;1:135–45.

[3] Janeway Jr CA, Medzhitov R. Innate immune recognition. Ann RevImmunol 2002;20:197–216.

[4] Hoffmann JA, Reichhart JM. Drosophila innate immunity: an evo-lutionary perspective. Nat Immunol 2002;3:121–6.

[5] Underhill DM. Toll-like receptors: networking for success. Eur JImmunol 2003;33:1767–75.

[6] Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol2003;21:335–76.

[7] Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homo-logue of the Drosophila Toll protein signals activation of adaptiveimmunity. Nature 1997;388:394–7.

[8] Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y,et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice arehyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lpsgene product. J Immunol 1999;162:3749–52.

[9] Lien E, Means TK, Heine H, Yoshimura A, Kusumoto S, FukaseK, et al. Toll-like receptor 4 imparts ligand-specific recognition ofbacterial lipopolysaccharide. J Clin Invest 2000;105:497–504.

10] Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. AToll-like receptor recognizes bacterial DNA. Nature 2000;408:740–5.

S2/18 G. Dougan, C. Hormaeche / Vaccine 24S2 (2006) S2/13–S2/19

[11] Takeshita F, Leifer CA, Gursel I, Ishii KJ, Takeshita S, Gursel M, etal. Cutting edge: role of Toll-like receptor 9 in CpG DNA-inducedactivation of human cells. J Immunol 2001;167:3555–8.

[12] Bazin H. The eradication of smallpox. New York: Academic Press;1999.

[13] Levine MM, Woodrow GC, Kaper JB, Cobon GS. New generationvaccines. New York: Marcel Dekker Inc.; 1997.

[14] Burdin M, Guy B, Moingeon P. Immunological foundations to thequest for new vaccine adjuvants. BioDrugs 2004;18:79–93.

[15] Kirk DD, Rempel R, Pinkhasov J, Walmsley AM. Application ofQuillaja saponaria extracts as oral adjuvants for plant-made vac-cines. Expert Opin Biol Ther 2004;4:947–58.

[16] Traub S, Kubasch N, Morath S, Kresse M, Hartung T, SchmidtRR, et al. Structural requirements of synthetic muropeptides to syn-ergize with lipopolysaccharide in cytokine induction. J Biol Chem2004;279:8694–700.

[17] Baldrick P, Richardson D, Wheeler AW, Woroniecki SR. Safety eval-uation of a new allergy vaccine containing the adjuvant monophos-phoryl lipid A (MPL((R))) for the treatment of grass pollen allergy.J Appl Toxicol 2004;24:261–8.

[18] Sato Y, Roman M, Tighe H, Lee D, Corr M, Nguyen MD, et al. Im-munostimulatory DNA sequences necessary for effective intradermalgene immunization. Science 1996;273:352–4.

[19] Girardin SE, Tournebize R, Mavris M, Page AL, Li X, Stark GR,et al. CARD4/Nod1 mediates NF-kappaB and JNK activation byinvasive Shigella flexneri. EMBO Rep 2001;2:736–42.

[20] Inohara N, Ogura Y, Chen FF, Muto A, Nunez G. Human Nod1confers responsiveness to bacterial lipopolysaccharides. J Biol Chem2001;276:2551–4.

[21] Girardin SE, Travassos LH, Herve M, Blanot D, Boneca IG, PhilpottDJ, et al. Peptidoglycan molecular requirements allowing detection

[

[

[

[

[[

[

[

[

[

[

[33] Tacket CO, Sztein MB, Wasserman SS, Losonsky G, Kotloff KL,Wyant TL, et al. Phase 2 clinical trial of attenuated Salmonellaenterica serovar Typhi oral live vector vaccine CVD 908-htrA inU.S. volunteers. Infect Immun 2000;68:1196–201.

[34] Hindle Z, Chatfield SN, Phillimore J, Bentley M, Johnson J, Cos-grove CA, et al. Characterization of Salmonella enterica derivativesharboring defined aroC and Salmonella Pathogenicity Island 2 TypeIII Secretion System (ssaV) mutations by immunization of healthyvolunteers. Infect Immun 2002;70:3457–67.

[35] Khan SA, Stratford R, Wu T, Mckelvie N, Bellaby T, HindleZ, et al. Salmonella typhi and Salmonella typhimurium deriva-tives harbouring deletions in aromatic biosynthesis and SalmonellaPathogenicity Island-2 (SPI-2) genes as vaccines and vectors. Vac-cine 2002;21:538–48.

[36] Sirard JC, Niedergang F, Kraehenbuhl JP. Live attenuatedSalmonella: a paradigm of mucosal vaccines. Immunol Rev1999;171:5–26.

[37] Monath TP, McCarthy K, Bedford P, Johnson CT, Nichols R, Yok-san S, et al. Clinical proof of principle for ChimeriVax: recombi-nant live attenuated vaccines against flavivirus infections. Vaccine2002;20:1004–18.

[38] Chabalgoity JA, Dougan G, Mastroeni P, Aspinall RJ. Live bacteriaas the basis for immunotherapies against cancer. Expert Rev Vaccines2002;1:495–505.

[39] Paglia P, Medina E, Arioli I, Guzman CA, Colombo MP. Genetransfer in dendritic cells, induced by oral DNA vaccination withSalmonella typhimurium, results in protective immunity against amurine fibrosarcoma. Blood 1998;92:3172–6.

[40] Medina E, Guzman CA, Staendner LH, Colombo MP, Paglia P.Salmonella vaccine carrier strains: effective delivery system to trig-ger anti-tumor immunity by oral route. Eur J Immunol 1999;29:693–

[

[

[

[

[

[

[

[

[

by Nod1 and Nod2. J Biol Chem 2003;278:41702–8.22] Dougan G. The molecular basis for the virulence of bacterial

pathogens: implications for oral vaccine development. Colworth Lec-ture. Microbiology 1994;140:215–24.

23] Sirard JC, Niedergang F, Kraehenbuhl JP. Live attenuatedSalmonella: a paradigm of mucosal vaccines. Immunol Rev1999;171:5–26.

24] Levine MM, Kaper JB, Herrington DA, Ketley J, Losonsky G,Tacket CO, et al. Safety, immunogenicity and efficacy of recom-binant live oral cholera vaccines. CVD 103 and CVD 103-HgR.Lancet 1988;2:467–70.

25] Mastroeni P, Chabalgoity JA, Dunstan SJ, Maskell DJ, Dougan G.Salmonella: immune responses and vaccines. Vet J 2001;161:132–64.

26] Moss B. Vaccinia virus vectors. Biotechnology 1992;20:345–62.27] Li A, Pal T, Forsum U, Lindberg AA. Safety and immunogenicity

of a live oral auxotrophic Shigella flexneri SFL124 in volunteers.Vaccine 1992;10:395–404.

28] Noriega FR, Wang JY, Losonsky G, Maneval DR, Hone DM, LevineMM. Construction and characterisation of a �elta aroA �elta virG S.flexneri 2a strain CVD1203, a prototype oral vaccine. Infect Immun1994;62:5168–72.

29] Kotfloff KL, Noriega F, Losonsky GA, Sztein MB, Wasserman SS,Nataro JP, et al. Safety, immunogenicity and transmissibility in hu-mans of CVD1203, a live oral Shigella flexneri 2a vaccine can-didate attenuated by deletions in aroA and virG. Infect Immun1996;64:4542–8.

30] Hoiseth K, Stocker BAD. Aromatic-dependant Salmonella ty-phimurium are non-virulent and effective as live vaccines. Nature1981;291:238–9.

31] Levine MM, Ferreccio Black CRE, Germanier R. Large-scale fieldtrial of Ty21a live oral typhoid vaccine in enteric-coated capsuleformulation. Lancet 1987;1:1049–52.

32] Tacket CO, Sztein MB, Losonsky GA, Wasserman SS, Nataro P,Edelman R, et al. Safety of live oral Salmonella typhi vaccine strainswith deletions in htrA and aroC aroD and immune response in hu-mans. Infect Immun 1997;65:452–6.

9.41] Sizemore DR, Branstrom AA, Sadoff JC. Attenuated bacteria as an

oral DNA delivery vehicle for DNA-mediated immunization. Science1995;270:299–302.

42] Darji CA, Guzman B, Gerstel P, Wachholz S, Timmis KN, WehlandJ, et al. Oral somatic transgene vaccination using attenuated S. ty-phimurium. Cell 1997;91:765–75.

43] Dietrich G, Bubert A, Gentschev I, Sokolovic Z, Simm A, Catic A,et al. Delivery of antigen-encoding plasmid DNA into the cytosolof macrophages by attenuated suicide Listeria monocytogenes. NatBiotechnol 1998;1:181–5.

44] Grillot-Courvalin C, Goussard S, Courvalin P. Wild-type intracel-lular bacteria deliver DNA into mammalian cells. Cell Microbiol2002;4:177–86.

45] Tacket CO, Kelly SM, Schodel F, Losonsky G, Nataro JP, EdelmanR, et al. Safety and immunogenicity in humans of an attenuatedSalmonella typhi vaccine vector strain expressing plasmid-encodedhepatitis B antigens stabilized by the asd-balanced lethal vector sys-tem. Infect Immun 1997;65:3381–5.

46] Gonzalez C, Hone D, Noriega FR, Tacket CO, Davis JR, Loson-sky G, et al. Salmonella typhi vaccine strain CVD 908 expressingthe circumsporozoite protein of Plasmodium falciparum: strain con-struction and safety and immunogenicity in humans. J Infect Dis1994;169:927–31.

47] Mollenkopf HJ, Groine-Triebkorn D, Andersen P, Hess J, KaufmannSH. Protective efficacy against tuberculosis of ESAT-6 secreted bya live Salmonella typhimurium vaccine carrier strain and expressedby naked DNA. Vaccine 2001;19:4028–35.

48] Nakayama K, Kelly SM, Curtiss III R. Construction of an Asd+ ex-pression cloning vector: stable maintenance and high level expres-sion of cloned genes in a Salmonella vaccine strain. BioTechnology1988;6:693–7.

49] Galen JE, Nair J, Wang JY, Wasserman SS, Tanner MK, Sztein MB,et al. Optimization of plasmid maintenance in the attenuated livevaccine vector strain Salmonella typhi CVD 908-htrA. Infect Immun1999;67:6424–33.

G. Dougan, C. Hormaeche / Vaccine 24S2 (2006) S2/13–S2/19 S2/19

[50] Strugnell RA, Maskell D, Fairweather N, Pickard D, CockayneA, Penn C, et al. Stable expression of foreign antigens fromthe chromosome of Salmonella typhimurium vaccine strains. Gene1990;88:57–63.

[51] Marshall D, Fowler R, Del Guidice G, Dorman C, Dougan G, BoweF. Use of the stationary phase inducible promoters, spv and dps, todrive heterologous antigen expression in Salmonella vaccine strains.Vaccine 2000;18:1298–306.

[52] Dunstan SJ, Simmons CP, Strugnell RA. Use of in vivo-regulatedpromoters to deliver antigens from attenuated Salmonella entericavar Typhimurium. Infect Immun 1999;67:5133–41.

[53] Stoute JA, Kester KE, Krzych U, Wellde BT, Hall T, White K, et al.Long-term efficacy and immune responses following immunizationwith the RTS, S malaria vaccine. J Infect Dis 1998;178(4):1139–44.

[54] Baldridge JR, McGowan P, Evans JT, Cluff C, Mossman S, JohnsonD, et al. Taking a Toll on human disease: Toll-like receptor 4 agonistsas vaccine adjuvants and monotherapeutic agents. Expert Opin BiolTher 2004;4:1129–38.

[55] Rappuoli R, Pizza M, Douce G, Dougan G. A relationship betweenthe structure and function of cholera and Escherichia coli heat labileenterotoxins and their immunological activity at mucosal surfaces.Immunol Today 1999;20:493–500.

[56] Douce G, Giannella V, Pizza M, Rappuoli R, Dougan G. Geneticallydetoxified mutants of Heat-Labile toxin of Escherichia coli are ableto act as oral adjuvants. Infect Immun 1999;67:4400–6.

[57] Freytag LC, Clements JD. Bacterial toxins as mucosal adjuvants.Curr Top Microbiol Immunol 1999;236:215–36.

[58] Fujihashi K, Koga T, van Ginkel FW, Hagiwara Y, McGhee JR.A dilemma for mucosal vaccination: efficacy versus toxicity usingenterotoxin-based adjuvants. Vaccine 2002;20:2431–8.

[59] Levine MM, Kaper JB, Black RE, Clements ML. New knowledgeon pathogenesis of bacterial enteric infections as applied to vaccinedevelopment. Microbiol Rev 1983;47(4):510–50.

[60] Dickinson BL, Clements JD. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity.Infect Immun 1995;63:1617–23.

[61] Giuliani MM, Del Giudice G, Giannelli V, Dougan G, Douce G,Rappuoli R, et al. Mucosal adjuvanticity and immunogenicity ofLTR72, a novel mutant of Escherichia coli heat-labile enterotoxinwith partial knockout of ADP-ribosyltransferase activity. J Exp Med1998;187:1123–32.

[62] De Magistris MT, Pizza M, Douce G, Ghiara P, Dougan G, Rap-puoli R. Adjuvant effect of non-toxic mutants of E. coli heat-labileenterotoxin following intranasal, oral and intravaginal immunization.Dev Biol Stand 1998;92:123–6.

[63] Yamamoto S, Takeda Y, Yamamoto M, Kurazono, Imaoka K, Ya-mamoto M, et al. Mutants in the ADP-ribosyltransferase cleft ofcholera toxin lack diarrheagenicity but retain adjuvanticity. J ExpMed 1997;185:1203–10.

[64] Agren LC, Ekman L, Lowenadler B, Lycke N. Genetically engi-neered nontoxic vaccine adjuvant that combines B cell targetingwith immunomodulation by cholera toxin A1 subunit. J Immunol1997;158:3936–46.

[65] Lycke N. From toxin to adjuvant: the rational design of a vaccineadjuvant vector, CTA1-DD/ISCOM. Cell Microbiol 2004;6(1):23–32.

[66] Mowat AM, Donachie AM, Jagewall S, Schon K, Lowenadler B,Dalsgaard K, et al. CTA1-DD-immune stimulating complexes: anovel, rationally designed combined mucosal vaccine adjuvant effec-tive with nanogram doses of antigen. J Immunol 2001;167:3398–405.