Download pdf - Recent developments in adjuvants for vaccines against infectious diseases

Biomolecular Engineering

18 (2001) 69–85

Review

Recent developments in adjuvants for vaccines againstinfectious diseases

Derek T. O’Hagan *, Mary Lee MacKichan, Manmohan SinghChiron Corporation, Immunology and Infectious Diseases, 4560 Horton Street, Emery�ille, CA 94608, USA

Abstract

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic thantraditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improvedvaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccinedelivery systems and ‘immunostimulatory adjuvants’. Vaccine delivery systems are generally particulate e.g. emulsions, micropar-ticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast,immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecularpatterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innateimmunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents havebeen combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatoryadjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses andthe ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development ofnew and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existingadjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIVwhich do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally. © 2001Published by Elsevier Science B.V.

Keywords: Adjuvants; Delivery systems; Vaccines; Cytokines; Immunostimulators

www.elsevier.com/locate/geneanabioeng

1. Introduction

Vaccines have traditionally consisted of live attenu-ated pathogens, whole inactivated organisms or inacti-vated toxins. In many cases, these approaches havebeen very successful at inducing immune protection,

mainly based on antibody responses. However, to de-velop vaccines against more ‘difficult’ pathogens, whichoften establish chronic infections, e.g. HIV, HCV, TBand malaria, the induction of cell mediated immunity(CMI) is likely to be necessary. Unfortunately, non-liv-ing vaccines have generally proven ineffective at induc-

Abbre�iations: APC, antigen presenting cells; CMI, cell mediated immunity; CMV, cytomegalovirus; CT, cholera toxin; CTB, cholera toxin Bsubunit; CTL, cytotoxic T lymphocyte; DC, dendritic cells; EBV, Ebola virus; FLUAD, adjuvanted influenza vaccine; HBV, hepatitis B virus;HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human pappiloma virus; HSV, herpes simplex virus; IFN-�, gammainterferon; IgE, immunoglobulin E; IgG, immunoglobulin G; IL-(2–12), interleukin (designated by the appropriate number, e.g. 2); IM,intramuscular; IN, intranasal; LPS, lipopolysaccharide; LT, labile enterotoxin; MALT, mucosal associated lymphoid tissue; MDP, muramyldipeptide; MHC II, major histocompatability complex class II; MHC, major histocompatability complex; MPL, monophosphoryl Lipid A; NKcells, natural killer cells; PAMP, pathogen associated molecular patterns; PLG, polylactide-co-glycolides; PRR, pattern recognition receptors; QuilA, Quillaja saponaria; SAF, syntex adjuvant formulation; SIV, simian immunodeficiency virus; T cells, T lymphocytes; TB, tuberculosis; Th1, Thelper 1; Th2, T helper 2; THD, toll homology domain; TLR, Toll-like receptors; TNF, tumor necrosis factor; VLP’s, virus-like particles.

* Corresponding author. Tel.: +1-510-923-7662; fax: +1-510-923-2586.E-mail address: derek–o’[email protected] (D.T. O’Hagan).

1050-3862/01/$ - see front matter © 2001 Published by Elsevier Science B.V.PII: S 1 3 8 9 -0344 (01 )00101 -0

D.T. O ’Hagan et al. / Biomolecular Engineering 18 (2001) 69–8570

ing CMI. In addition, although live vaccines may in-duce CMI, some live attenuated vaccines can causedisease in immunosuppressed individuals and somepathogens are difficult or impossible to grow in culture(e.g. HCV). Moreover, many traditional inactivatedvaccines (e.g. Bordetella pertussis) also contain compo-nents that can cause undesirable effects and safetyproblems. As a result of these problems, several newapproaches to vaccine development have emerged,which may have significant advantages over more tradi-tional approaches. These approaches include: (1) re-combinant protein subunits; (2) synthetic peptides; (3)protein polysaccharide conjugates; and (4) plasmidDNA. While these new approaches may offer impor-tant safety advantages, a general problem is that thevaccines alone are often poorly immunogenic. Tradi-tional vaccines contain many components, some ofwhich can elicit additional T cell help or function asadjuvants, e.g. bacterial DNA in whole cell vaccines.However, these components have been eliminated frommany new generation vaccines. Therefore, there is anurgent need for the development of potent and safeadjuvants that can be used with newer generation vac-cines, including DNA vaccines. In the recent years therehas been great interest in DNA vaccines, since theyappear to offer significant potential for the induction ofpotent CMI [1], which has been very difficult to achievewith non-living vaccines. Nevertheless, the potency ofDNA vaccines in humans has so far been disappoint-ing, particularly in relation to their ability to inducehumoral responses [2,3]. This has prompted investiga-tors to work both on adjuvants and delivery systems forDNA vaccines [4] and also to use DNA in a prime/boost setting with alternative modalities, e.g. liveviruses [5–7].

Immunological adjuvants were originally describedby Ramon [8] as ‘substances used in combination witha specific antigen that produced a more robust immuneresponse than the antigen alone’. This broad definitionencompasses a very wide range of materials [9]. How-ever, despite extensive evaluation of a large number ofcandidates over many years, the only adjuvants cur-rently approved by the U.S. Food and Drug Adminis-tration are aluminum based mineral salts (genericallycalled alum). Alum has a good safety record, butcomparative studies show that it is a weak adjuvant forantibody induction to protein subunits and a pooradjuvant for CMI [10]. Moreover, alum adjuvants caninduce IgE antibody responses and have been associ-ated with allergic reactions in some subjects [10,11].Although Alum has been used as an adjuvant for manyyears, its mechanism of action remains poorly defined.It was originally thought to provide a ‘depot’ effect,resulting in persistence of antigen at the injection site.However, more recent studies involving radio-labelledantigens have shown that Alum does not establish an

antigen depot at the injection site [12]. Recent work invitro has indicated that Alum upregulates co-stimula-tory signals on human monocytes and promotes therelease of IL-4 [13]. Alum adsorption may also con-tribute to a reduction in toxicity for some vaccines, dueto the adsorption of contaminating endotoxin [14].

A key issue in adjuvant development is toxicity, sincesafety concerns have restricted the development of ad-juvants since Alum was first introduced more than 50years ago [15]. Many experimental adjuvants have ad-vanced to clinical trials and some have demonstratedhigh potency, but most have proven too toxic forroutine clinical use. For standard prophylactic immu-nization in healthy individuals, only adjuvants thatinduce minimal adverse effects will prove acceptable. Incontrast, for adjuvants which are designed to be used inlife-threatening situations e.g. cancer vaccines, the ac-ceptable level of adverse events would likely be in-creased. This review will focus predominantly onadjuvants to be used in vaccines against infectiousdiseases, although the potential use of similar adjuvantsin cancer vaccines will be covered when appropriate.Developments in cancer vaccines have recently beenreviewed [16]. There has been much concern recentlythat potent adjuvants might activate immunity to suchan extent that auto-immune conditions might be trig-gered. This might be a particular concern for adjuvantswhich mimic components of pathogenic microorgan-isms and provide potent pro-inflammatory signals.Clearly, the timing and localization of certain stimulimay prove to be important in this context and limitingdistribution of adjuvant actives to key cells is likely tobe beneficial. To date, autoimmunity has only beenlinked to immunization in exceptional cases. Neverthe-less, as more potent adjuvant actives become available,particularly those which activate innate immune re-sponses, this needs to be monitored closely. Additionalpractical issues which are important for adjuvant devel-opment include biodegradability, stability, ease of man-ufacture, cost and applicability to a wide range ofvaccines. Ideally, for ease of administration and en-hanced patient compliance, the adjuvant should allowthe vaccine to be administered by a mucosal route, butthis has proven difficult. Examples of some of theadjuvants that have been evaluated in clinical trials areshown in Table 1. Although the mechanisms of actionof adjuvants often remain poorly understood [9,15],there is currently great interest in the effects of adju-vants on non-specific, or innate immunity.

2. The role of adjuvants in vaccine development

Adjuvants can be used to improve the immune re-sponse to vaccine antigens in several different ways: (1)adjuvants can increase the immunogenicity of weak

D.T. O ’Hagan et al. / Biomolecular Engineering 18 (2001) 69–85 71

antigens; (2) enhance the speed and duration of theimmune response; (3) modulate antibody avidity, spe-cificity, isotype or subclass distribution; (4) stimulatecell mediated immunity (CMI), (5) promote the induc-tion of mucosal immunity; (6) enhance immune re-sponses in immunologically immature, or senescentindividuals; (7) decrease the dose of antigen in thevaccine and reduce costs; or (8) help to overcomeantigen competition in combination vaccines.

The mechanisms of action of most adjuvants stillremain only poorly understood, and this has impededthe rational development of new adjuvants. Immuniza-tion often activates a complex cascade of responses,and the primary effect of the adjuvant is often difficultto clearly discern in vivo. However, if one accepts therecently proposed geographical concept of immune re-activity, in which antigens which do not reach the locallymph nodes do not induce responses [17], it becomeseasier to propose mechanistic interpretations of theimportant effects of some adjuvants. If antigens whichdo not reach lymph nodes do not induce responses,then any adjuvant which enhances delivery of the anti-gen to the node may enhance the response. Antigendelivery may be enhanced in several ways; the adjuvant

may increase cellular infiltration into the injection site,so that more cells are present to take up antigen, it maydirectly promote the uptake of antigen into antigenpresenting cells (APC), or it may directly contribute tothe delivery of antigen to the local lymph nodes. Themost important APC involved in antigen capture arethe dendritic cells (DC), which in their immature stateact as ‘sentinels’ and circulate through peripheral tis-sues. Following uptake of antigen and cell activation,DC undergo maturation and migrate to lymph nodes,where they have the unique capacity to present antigensto naı̈ve T cells. Hence, the principal mode of action ofa range of particulate adjuvants, or ‘antigen deliverysystems’ e.g. microparticles, emulsions, liposomes, Is-coms etc. may be to promote uptake of the antigen byAPC at the injection site. However, some of thesedelivery systems may also be capable of moving awayfrom the injection site in lymph and may deliver anti-gen directly to the lymph node. Nevertheless, the suc-cessful delivery of antigen to a lymph node will notnecessarily result in the induction of an immune re-sponse, since the presence of antigen alone constitutesonly ‘signal 1’. To successfully induce an immune re-sponse, it is necessary that ‘signal 2’ is also present.Signal 2 is represented by co-stimulatory molecules andcytokines, which are normally provided by APC andcontribute to the priming of T helper cells. The role ofthe T helper cells is to provide antigen specific help forB cell proliferation and antibody induction, and helpfor cytotoxic T lymphocyte (CTL) responses. In theabsence of signal 2, non-responsiveness, or ‘immuno-logical tolerance’ will result. In addition to adjuvantswhich act predominantly as delivery systems, facilitat-ing antigen uptake, transport or presentation by APC,there is a second broad class of adjuvants which arethought to directly activate signal 2. This is achievedthrough stimulating the release of cytokines, or theexpression of co-stimulatory molecules on APC. Manyof these adjuvants are directly derived from pathogense.g. bacterial cell wall components, and may be per-ceived as a ‘danger signal’ by the innate immune sys-tem, indicating possible infection of the host. It hasbeen proposed that agents which activate innate immu-nity should be referred to as possessing pathogen asso-ciated molecular patterns (PAMP’s), which interactwith their cognate binding partners on phagocytes,called pattern recognition receptors (PRR) [18]. Animportant consequence of the interaction of bacterialcell wall components with PRR on cells of the innateimmune system is the activation of pro-inflammatorypathways, which direct the induction of acquired im-munity [19]. Nevertheless, the induction of immunity isnot necessarily dependent on the presence of exogenouspathogen derived materials. It has been shown thatpotent endogenous adjuvants can be released fromstressed or dying cells [20] and by cells undergoing

Table 1Selected examples of vaccine adjuvants

Mineral salts Aluminum hydroxide*Aluminum phosphate*Calcium phosphate*

Immunostimulatory adjuvants Cytokines e.g. IL-2, IL-12,GM-CSF

Saponins e.g. QS21MDP derivativesBacterial DNA (CpG oligo’s)LPSMPL and synthetic derivativesLipopeptides

Lipid particles Emulsions e.g. Freund’s, SAF,MF59*

LiposomesVirosomes*Iscom’sCochleates

Particulate adjuvants PLG microparticlesPoloxamer particlesVirus-like particles

Mucosal adjuvants Heat labile enterotoxin (LT)Cholera toxin (CT)Mutant toxins e.g. LTK63 and

LTR72MicroparticlesPolymerized liposomesChitosan

With the exception of cochleates and polymerized liposomes, all ofthese adjuvants have been evaluated in clinical trials. However, onlythose marked * are currently included as adjuvants in approvedvaccine products.


Table 2Functional elements of the immune response

Response/mediator Function

Take up, process and presentAPCantigens as peptides throughMHC I and II pathways,provide co-stimulatory signals.DC have unique ability topresent antigen to naı̈ve T cells

Antibody production and APCB lymphocytesfunction

T lymphocytesCD8+(MHC I) CTL—Kill infected cells,

secrete cytokines that suppressviral replicationPromote B cell and CTLCD4+(MHC II)differentiation and maturationSecrete IFN-�, mediates killingTh1of intracellular pathogens

Th2 Secrete IL-4, provides help forantibody induction

Co-stimulatory signals e.g. B7, Necessary for effective antigenCD40, CD28 presentation and T cell

activation

Promote cytokine induction orAdjuvantsco-stimulatory signals orenhance antigen uptake byAPC’s

most important for prevention of infection, but thismight be very difficult to achieve with some pathogense.g. HIV. In contrast, T cells are mainly responsible forrecognizing and killing infected cells and function tolimit the spread of infection. For most pathogens, theinitial infections can be successfully eradicated by theimmune response, or the infection is inherently self-lim-iting. However, some pathogens are successful at estab-lishing a reservoir of infection that is very difficult toclear e.g. HIV and HCV. Potent adjuvants and novelvaccine strategies may be required to allow the success-ful elimination of these reservoirs.

The dominant paradigm in immunology for severaldecades was that the immune system evolved to dis-criminate self from nonself [23]. This hypothesis re-sulted in significant progress in understanding theclonal recognition of antigenic epitopes mediated by Band T lymphocytes. However, the self/nonself frame-work offers little insight into why some nonself antigensare found to be poorly immunogenic. In the lastdecade, alternative models of immunity have been es-tablished, which emphasize the selective pressures onthe host to induce a pro-inflammatory innate immuneresponse to PAMP’s [24,25] and tissue damage [26–28].These more ancient responses are not antigen-specificand are mediated by the innate immune system [29,30].In this new model of immunity, vaccines will elicit apotent immune response only when the nonself antigensmimic key aspects of infectious agents or cause somedegree of localized tissue damage.

Traditional vaccines such as bacterial toxoids andattenuated viral vaccines often contain all or most ofthe features of real pathogens and therefore, are suffi-ciently potent to induce protective immune responses.In contrast, recombinant vaccines are highly purified,lack many of the features of the original pathogen anddo not evoke strong immune responses. Therefore,vaccine adjuvants are crucial to make the vaccine suffi-ciently immunogenic. Hence it can be argued that therole of vaccine adjuvants for recombinant vaccines is toensure that the vaccine resembles infection closelyenough to initiate a potent immune response [24,29]. Inaddition, the innate immune system directs the balanceof humoral and CMI [30], and adjuvants can controlthe type of acquired immune response induced [31].

4. Cell types and effector mechanisms of innateimmunity

Initiation of an immune response by vaccinationrelies on innate immune cells, and is mediated in largepart by neutrophils and macrophages. These cellsphagocytose and kill pathogens, but additionally co-or-dinate the adaptive response by secreting a range ofinflammatory mediators and cytokines. Adjuvants can

apoptosis [21]. In addition, it has been demonstratedthat DC acquire antigen from apoptotic cells, and thatthis may help to explain tumor immunogenicity andtransplant rejection [22]. For the purposes of this re-view, PAMP’s will be referred to as ‘immunostimula-tory’ adjuvants, to differentiate them from adjuvantswhich work predominantly as antigen delivery systems.

The most appropriate adjuvant to use for a givenvaccine antigen will depend to a large extent on thetype of immune response that is required for protectiveimmunity. However, for many infectious diseases, thecorrelates of protective immunity have yet to be estab-lished. Therefore, in many situations, adjuvant selectionremains rather empirical.

3. An immunologic perspective on adjuvants

Some of the key components involved in inductionand maintenance of an immune response are summa-rized briefly in Table 2. Immune protection followingvaccination depends predominantly on the generationof immunologic memory, mediated by B and Tlymphocytes of the acquired immune system, whichhave highly restricted antigen specificity. Vaccines areeffective either due to prevention of infection, or toprevention of disease. Antibodies are thought to be


elicit cytokine and chemokine production by APC, theycan recruit cells to the local tissue and node, and candirect the development of humoral and CMI.

When APC are activated they become more potentactivators of T cells by virtue of upregulation of MHCand costimulatory molecules on their surface [32]. Acti-vated DC (also called mature DC) are the only APCcapable of effectively activating naı̈ve T cells, clearly adesirable outcome for vaccination against many patho-gens, especially viruses. While less is understood on theroles of lymphocytes (B, T, and NK cells) in innateimmunity, polyclonal activation of these cells indepen-dent of antigen recognition is possible and likely con-tributes to the activity of some adjuvants, e.g. MPL[33].

Although not all of the features that make pathogensimmunogenic are well understood, certain components,including PAMP’s, clearly have the ability to activateAPC and initiate immune responses. DC have beenshown to be activated by diverse microbes and theircharacteristic structures (reviewed in [34]), by lipo-polysaccharide (LPS), which is the prototypic activatorof innate immunity [35,36], and by endogenous cytoki-nes, e.g. TNF [36]. APC activation by exposure tobacteria depends on engagement of PRR and activationof signals that alter gene expression [37–39]. Thesereceptors and signals are not completely understood.However, the Toll receptor family, originally describedin Drosophila [40], has recently emerged as a key playerin pathogen recognition and may be a good target forrational adjuvant development [19].

Toll-like receptors (TLR) mediate responses of mam-malian APC to pathogens and their characteristic struc-tures (PAMP’s), including LPS. TLR signal via aconserved intracellular motif, the Toll homology do-main (THD). THD structure and its function in patho-gen recognition and response initiation have beenevolutionarily conserved across plants, flies and mam-mals (reviewed in [41]). In Drosophila, there is evidencethat TLR mediate responses to bacteria and fungi, andthat different Toll receptor family members are acti-vated by different pathogen structures and initiate over-lapping yet distinct responses [42,43]. At present, atleast 10 mammalian TLR have been identified and havebeen shown to be required for responses to cell-wallcomponents of Gram-negative and -positive bacteria,mycobacteria and fungi [19,44]. Activation of murinecells by LPS requires the presence of TLR4, and LPS-hyporesponsive mouse strains lack the wildtype recep-tor [45]. In human cells, TLR4 can mediate responsesto LPS in vitro [46] and in vivo [47]. Exposure to LPSinduces activation of MAP kinase pathways and NF-�B, which in turn regulate gene expression of pro-infl-ammatory cytokines and chemokines [48]. TLR4 is alsothe ligand for human heat shock protein 60, which isreleased from stressed or damaged cells and elicits a

potent pro-inflammatory response of the innate im-mune system [49]. TLR2 mediates responses to peptido-glycan, zymosan and microbial lipoproteins [50,51],while TLR6 co-operates with TLR2 in detecting asubset of bacterial peptidoglycans [52]. Indeed, it ap-pears that the various TLR may co-operate to providea combinatorial repertoire to recognize a wide range ofbacterially derived products. The TLR may functionthrough different intracellular signaling proteins, tobring about different types of response. Current evi-dence suggests that several adjuvants exert their actionsvia mammalian Toll-like receptors. One class of im-munostimulatory adjuvants are derived from LPS ofgram-negative bacteria. The most extensively evaluatedmember of this family, monophosphoryl Lipid A(MPL), is obtained from Salmonella minnesota. Re-sponses to a variety of mycobacterial components havebeen shown to require TLR2 in mouse and human cells[53,54]. Thus part of the activity of complete Freund’sadjuvant, which contains mycobacterial products, islikely to be mediated by TLR2. In Drosophila, Toll and18-wheeler, a Toll-related receptor, are activated byfungi and bacteria, respectively, inducing expression ofdifferent subsets of antimicrobial peptides [41]. By anal-ogy, it may 1 day be possible to evoke the immuneresponse profile of choice, e.g. Th1 vs. Th2 (see below),by targeted activation of a particular mammalian TLRtype. Hence it appears that innate immunity may not beas non-specific as originally thought. Interestingly, themammalian receptors for the cytokines IL-1 and IL-18,like TLRs, signal via THDs and influence immuneresponses.

5. Induction of acquired cellular immunity

CD4+T cells recognize antigens after they havebeen processed through the exogenous pathway byAPC, expressing major histocompatability complexclass II (MHC II) molecules. After activation, CD4+Tcells differentiate into functional subsets, termed Thelper 1 (Th1) and Th2. The division into these subsetsis based largely on their secretion of different cytokines[55–57]. Th1 responses are typically characterized bythe secretion of IFN-� and the generation of delayedtype-hypersensitivity responses. Th1 cells are thought tomediate the killing of intracellular pathogens, mainlythrough IFN-�. The production of Th1 responses ap-pears to be crucially dependent on IL-12 production,which is produced by phagocytic APC, following expo-sure to pathogens. In contrast, Th2 responses are char-acterized by the secretion of IL-4, IL-5, IL-6 and IL-10,and help in the induction of high levels of circulatingantibodies. The different cytokine secretion pathwaysappear to be mutually antagonistic to some extent, andupregulation of one type of response normally results in


down-regulation of the alternative. In mice, the produc-tion of the IgG2a antibody isotype is widely recognizedas characteristic of a Th1 response [58], while a Th2response is associated with the induction of IgG1.There are strong indications that cytokines produced byDC following their activation or maturation directlyinfluence T helper cytokine profiles, i.e. the develop-ment of Th1 vs. Th2 responses [59]. Although differentadjuvants may induce comparable levels of functionalantibodies, the respective cytokine profiles and anti-body isotypes may differ (Fig. 1a–c). In certain situa-tions, the type of response induced (Th1 or Th2), mayhave a significant impact on the protective efficacy of avaccine. Different adjuvants tend to favor specific types

of responses and this may provide some degree ofguidance in adjuvant selection. However, adjuvantchoice is often complicated by commercial constraints,toxicity and availability.

CD8+T cells recognize antigens that are processedendogenously and presented by cells expressing MHCclass I molecules. CD8+T cells mediate their effectorfunction through the production of cytokines, such asIFN-� and TNF-�, and through a direct cytolytic ef-fect. The cytolytic effect of CD8+CTL is mediated bythe release of granule contents, such as perforin andgranzyme. In addition, CD8+CTL can kill by a pro-cess of Fas-mediated lysis.

6. Immunostimulatory adjuvants

As discussed earlier, MPL adjuvant is a PAMPwhich is derived from bacterial cell walls and interactswith TRL4. In a number of pre-clinical studies, MPLhas been shown to induce the synthesis and release ofcytokines, particularly IL-2 and IFN-�, which promotesthe generation of Th1 responses [60,61]. In addition,MPL appears to increase the migration and maturationof DC [62]. MPL as a single adjuvant does not appearto be very potent for antibody induction, although itappears effective for the induction of CD4+T cellresponses. Often, MPL has been formulated into emul-sions to promote its potency [63]. Clinically, MPL hasoften been used in complex formulations, includingliposomes and emulsions, and has also been used inadjuvant combinations with alum and QS21, whichmakes it difficult to determine the contribution of MPLto the overall adjuvant effect. For example, MPLshowed good tolerability and an adjuvant effect in alimited number of volunteers in combination with alum[64]. MPL has been extensively evaluated in the clinic,�10 000 subjects immunized (T. Ulrich, personal com-munication) for cancer (melanoma and breast) andinfectious disease vaccines (genital herpes, HBV,malaria and HPV), and for allergies, with an acceptableprofile of adverse effects. Although not yet included inan approved vaccine product, phase III trials are under-way with MPL for infectious diseases and it is used inGermany on a named patient basis for allergy vaccines(Allan Wheeler, personal communication). In addition,structure-function studies of MPL allowed identifica-tion of a new generation of synthetic adjuvants calledAGPs [65], the lead candidate of which (Ribi. 529), iscurrently being evaluated in a phase III trial for HBV.It has also been claimed that MPL may be used as anadjuvant for DNA vaccines [66] and for mucosal deliv-ery of vaccines [67].

In the last few years, a whole new class of adjuvantactives have been identified. This followed from thedemonstration that bacterial DNA, but not vertebrate

Fig. 1. (a) Herpes simplex virus type 2 neutralizing titers in twogroups of mice (n=10) immunized (day 0 and 28) with 10 �g ofrecombinant gD2 in MF59 or entrapped in PLG microparticles. (b)Serum IgG antibody isotypes (IgG2a and IgG1) in two groups ofmice (n=10) immunized (days 0, 7 and 14) with 10 �g recombinantgD2 in MF59 and PLG microparticles. Geometric mean titer�S.E.is represented. (c) Serum cytokine profiles (IL-4, IL-5 and IFN-�)from two groups of mice (n=10) immunized (days 0, 7 and 14) with10 �g recombinant gD2 in MF59 or PLG microparticles.


DNA, had direct immunostimulatory effects on im-mune cells in vitro [68,69]. The immunostimulatoryeffect was due to the presence of unmethylated CpGdinucleotides [70], which are under-represented andmethylated in vertebrate DNA. Unmethylated CpG inthe context of selective flanking sequences are thoughtto be recognized by cells of the immune system to allowdiscrimination of pathogen-derived DNA from selfDNA [71]. Bacterial DNA containing CpG oligonucle-otides activate cells of the innate immune system, in-cluding macrophages and DC’s, to upregulate MHCclass II and co-stimulatory molecules, to transcribecytokine mRNAs and to secrete pro-inflammatory cy-tokines [72]. It has recently been shown that cellularresponses to CpG DNA is mediated by binding toTLR9 [73]. Previously, it was reported that CpG aretaken up by non-specific endocytosis and that endoso-mal maturation is necessary for the activation of stresskinase pathways, resulting in the release of pro-inflam-matory cytokines [74]. Hence, the localization of TLR9awaits confirmation and the mechanism of cellular up-take and activation for CpG containing DNA remainsto be more fully defined. However, it appears likely thatTLR are recruited to phagosomes of innate immunecells, where they sample the contents, with specific TLRresponding to specific microbial products.

Exposure to CpG motifs brings about conversion ofimmature DC to mature APC [75]. CpG motif’s aremost potent for the induction of Th1 responses, mainlythrough stimulating TNF�, IL-1, IL-6 and IL-12, andthrough the expression of co-stimulatory molecules[75,76]. CpG also appear to have significant potential asmucosally administered adjuvants [77,78]. The adjuvanteffect of CpG appears to be maximized by their conju-gation to protein antigens [79], or their formulationwith delivery systems [218]. Importantly, CpGs alsoappear to have significant potential for the modulationof existing immune responses, which may be useful invarious clinical settings, including allergies [80]. Al-though, CpG oligo’s have mainly been evaluated inrodent models and with murine cells, recent papershave begun to describe sequences that are active inprimates, including humans [81]. Moreover, recent re-ports have also indicated that potent adjuvant affectshave been achieved in human clinical trials (HeatherDavis, unpublished observations). Although, it is tooearly to know in which situations CpG oligo’s mightprove to be most advantageous, their apparent abilityto selectively manipulate Th1 responses is most excit-ing. Nevertheless, the safety of CpG DNA needs to befirmly established in the clinic, since the induction ofautoimmunity with CpG immunization can be shownin various established animals models [82]. However,the relevance of these observations to human studies isunknown.

A third group of immunostimulatory adjuvants arethe triterpenoid glycosides, or saponins, derived fromthe bark of a Chilean tree, Quillaja saponaria. Al-though not actually pathogen derived and therefore nota PAMP, saponins function mainly through the induc-tion of cytokines and may prove to have interactionswith PRR. Saponins have been widely used as adju-vants for many years and have been included in veteri-nary vaccines. However, saponins are surface activeagents and cause haemolysis of red blood cells in vitro,although haemolysis does not appear to correlate withadjuvant activity [83]. A pure fraction of Quil A sa-ponin with low toxicity (QS21) was isolated by Kensil,who defined the structural moieties responsible for theadjuvant activity [84]. QS21 has been shown to be apotent adjuvant for CTL induction, and induces Th1cytokines (IL-2 and IFN-�) and antibodies of theIgG2a isotype [83]. Saponins have been shown to inter-calate into cell membranes, through interaction withcholesterol, forming ‘holes’ or pores [85]. Although it isunknown if the adjuvant effect of saponins is related topore formation, this may allow antigens to gain accessto the endogenous pathway of antigen presentation, forCTL induction. A number of clinical trials have beenperformed, using QS21 as an adjuvant, initially forcancer vaccines (melanoma, breast and prostate), andsubsequently for infectious diseases, including HIV-1,influenza, herpes, malaria and hepatitis B [86]. Doses of200 �g or higher of QS21 have been associated withsignificant local reactions [86], but lower doses appearto be better tolerated. More than 1600 volunteers havebeen immunized with QS21 containing vaccines and themost common complaint is of pain/tenderness at theinjection site, which is dose dependent and usually ofshort duration [86]. Nevertheless, a review of 25 inde-pendent clinical trials for HIV vaccines concluded thatthree adjuvants, including an MDP derivative andQS21 were associated with moderate to severe localreactions [87]. In a recent clinical trial with HIV-1 envantigen, QS21 was able to allow a significant dosereduction for the antigen and also enhanced prolifera-tive T cell responses, but not CTL [88]. However, painon injection was a common problem for many vaccinerecipients. Hence the balance of potency vs. adverseevents is key for this adjuvant and an effective adjuvantdose which is tolerable needs to be established in hu-mans for each vaccine indication. QS21 has also beenclaimed to perform as an adjuvant for DNA vaccines,following both systemic and mucosal administration[89].

As an alternative to the use of cytokine inducingadjuvants, cytokines may also be used directly. Mostcytokines have the ability to modify and re-direct theimmune response. The cytokines which have been eval-uated most extensively as adjuvants include IL-1, IL-2,IFN-�, IL-12 and GM-CSF [90]. However, all of these


Table 3A comparison of the relative dimensions of pathogens and particulateadjuvants

Particulate adjuvantsNatural pathogens

�0.5–3 �mBacteria Microparticles 100 nm–10 �m�250 nm LiposomesPoxvirus 50 nm–10 �m

virosomesherpesvirusHIV Influenza �100 nm MF59 �200 nm

virus20–30 nm Iscoms �40 nmPoliovirus

parvovirusVirus-like �20–50 nmparticles

the presence of additional immunostimulatory adju-vants, which proved to be a potent adjuvant with anacceptable safety profile [94]. MF59 enhanced the im-munogenicity of influenza vaccine in small animal mod-els [95–97] and was shown to be a more potentadjuvant than alum for hepatitis B vaccine (HBV) inbaboons [98]. Subsequently, the safety and immuno-genicity of MF59 adjuvanted influenza vaccine(FLUAD)™ was confirmed in elderly subjects in clini-cal trials [99,100] and this data allowed the approval ofthis product for licensure in Italy in 1997, and forseveral additional countries through mutual recognitionin 2000. The potency of MF59 as an adjuvant forinfluenza vaccines may be particularly advantageous inthe face of a potential pandemic [219]. The potency ofMF59 for HBV has also been confirmed in a humanclinical trial, in which MF59 was shown to be 100-foldmore potent than the commercial Alum adjuvantedvaccine [101]. In addition, MF59 has also been shownto be an effective adjuvant for a protein/polysaccharideconjugate in infant baboons [102]. Studies with labelledMF59 have shown that it is taken up by macrophagesand DC, both at the site of injection and in local lymphnodes [103]. A recent paper has suggested that someparticulate adjuvants, including squalene emulsions,may exert their adjuvant effect partly by inducingmacrophage survival and an enhanced proliferative re-sponse to GM-CSF and CSF-1 [104]. Experience in theclinic (�18 000 subjects immunized in Chiron con-trolled clinical trials), with HIV, HSV, CMV, HBV andinfluenza, has shown that MF59 is safe and well toler-ated in humans [105–108] In addition, MF59 wasshown to be safe and well tolerated in newborn infantsin a HIV vaccine trial [109]. Moreover, MF59 can beused with recombinant proteins as an effective boostervaccine in individuals primed with a live virus vaccine[110]. In summary, MF59 is a safe and well-toleratedadjuvant in humans and is effective for the induction ofpotent antibody responses.

In some studies, emulsions have also been used asdelivery systems for immunostimulatory adjuvants, in-cluding MPL and QS21. This approach allows im-munostimulatory adjuvants to be targeted for enhanceduptake by APC. The level of protection induced in amouse model of malaria by this adjuvant formulationwas comparable or better than the levels of protectioninduced with the vaccine in Freund’s complete adjuvant[111]. The adjuvant formulation (SBAS-2) subsequentlyshowed protective efficacy against an experimentalchallenge in human volunteers exposed to infectedmosquitoes, although protection was of short duration[112]. Although SBAS-2 induces potent antibody andTh1 responses, its toxicity in humans remains to befurther evaluated. In a subsequent trial with HIV-1 env,SBAS-2 induced high titers and proliferative T cellresponses, but did not induce CTL or primary isolate

molecules exhibit dose related toxicity. In addition,since they are proteins, they have stability problems, ashort in vivo half-life and are relatively expensive.Therefore, it is unlikely that cytokines will prove ac-ceptable for use as adjuvants in routine vaccination.Nevertheless, considerable progress has been made inthe use of cytokines for the immunotherapy of cancer[91].

7. Particulate antigen delivery systems

The use of particulate adjuvants, or antigen deliverysystems, as alternatives to immunostimulatory adju-vants has been evaluated by several groups. Particulateadjuvants (e.g. emulsions, microparticles, iscoms, lipo-somes, virosomes and virus-like particles) have com-parable dimensions to the pathogens which the immunesystem evolved to combat (Table 3). Immunostimula-tory adjuvants may also be included in particulatedelivery systems to enhance the level of response, or tofocus the response through a desired pathway, e.g. Th1or Th2. In addition, formulating potent immunostimu-latory adjuvants into delivery systems may limit adverseevents, through restricting the systemic circulation ofthe adjuvant.

8. Lipid particles as adjuvants

A potent oil-in-water (o/w) adjuvant, the syntex adju-vant formulation (SAF) [92] was developed using abiodegradable oil (squalane) in the 1980s, as a replace-ment for Freund’s adjuvants. Freund’s adjuvants arepotent but toxic water in mineral oil adjuvants, whichmay also contain killed mycobacteria [93]. However,SAF contained a bacterial cell wall based syntheticadjuvant, threonyl muramyl dipeptide (MDP), and anon-ionic surfactant, poloxamer L121, and proved tootoxic for widespread use in humans [15]. Therefore, asqualane o/w emulsion was developed (MF59), without


neutralizing antibodies [113]. Nevertheless, this formu-lation was associated with a significant number ofadverse events and the reactogenicity profile observedappeared to preclude its use for most if not all prophy-lactic vaccines. An alternative approach involves theuse of the Montanide series of adjuvants, which areformulated as water in oil emulsions [114]. Althoughpotent for antibody induction, these adjuvants arelikely to prove too toxic for routine clinical use.

Liposomes are phospholipid vesicles which have beenevaluated both as adjuvants and as delivery systems forantigens and adjuvants [115,116]. However, liposomeshave been commonly used in complex adjuvant formu-lations, often including MPL, which makes it difficultto determine the contribution of the liposome to theoverall adjuvant effect. Nevertheless, several liposomalvaccines based on viral membrane proteins (virosomes)without additional immunostimulators, have been ex-tensively evaluated in the clinic and are approved asproducts in Europe for hepatitis A and influenza [117].Modified liposomal structures termed ‘cochleates’ arealso being evaluated as systemic and mucosal adjuvantsin small animal models [118]. In addition, the recentdescription of liposomes prepared with cationic lipidsto which antigens may be adsorbed appears promising[119]. The development of polymerized liposomes,which show enhanced stability in the gut, also offerspotential for the development of mucosal vaccines[120].

The immunostimulatory fractions from Quillaja sa-ponaria (Quil A) have been incorporated into lipidparticles comprising cholesterol, phospholipids and cellmembrane antigens, which are called iscoms [121]. Themechanism of action of the adjuvant Quil A is thoughtto be very similar to QS21, which is a purified singlefraction from Quil A. In a study in macaques, aninfluenza iscom vaccine was shown to be more im-munogenic than a classical subunit vaccine and inducedenhanced protective efficacy [122]. A similar formula-tion has been evaluated in human clinical trials and hasbeen shown to induce CTL responses [123]. The princi-pal advantage of the preparation of iscoms is to allowa reduction in the dose of the hemolytic Quil A adju-vant and to target the formulation directly to APC’s. Inaddition, within the Iscom structure, the Quil A isbound to cholesterol and is not free to interact with cellmembranes. Therefore, the hemolytic activity of thesaponins is significantly reduced [84,121]. It is wellestablished that Iscoms induce cytokine production in arange of mouse strains and a recent study has indicatedthat the induction of IL-12 is key to the adjuvant effectof iscoms [124], although in previous studies, strongIFN-� responses were induced [125]. In a study inrhesus macaques, iscoms induced potent Th1 responsesagainst HIV-1 env, while MF59 induced more of a Th2response, but both vaccines offered a significant degree

of protection against viral challenge [126]. Althoughnot evaluated in this study, iscoms are generally consid-ered to be the most potent adjuvant for the induction ofCTL responses with recombinant proteins in pre-clini-cal models. In a recent study, we demonstrated theinduction of potent long lasting CTL responses inrhesus macaques immunized with core from hepatitis Cvirus adsorbed to a novel iscom formulation [127]. Inaddition, potent T cell proliferative responses have beeninduced in primates with iscom vaccines containingCMV, flu, HIV and EBV antigens [121,128]. However,the efficacy for CTL induction, and the safety profile ofiscoms needs to be further established in human sub-jects, although initial studies are encouraging [129].Nevertheless, a potential problem with iscom’s is thatinclusion of antigens into the adjuvant is often difficult,and may require extensive antigen modification [130].Nevertheless, recent work has identified novel ways bywhich some antigens can be effectively associated withiscoms, without significant formulation difficulties[127].

An alternative lipid vehicle approach has been de-scribed involving non-ionic surfactant vesicle, or ‘nio-somes’, which have induced potent responses in smallanimal models [131]. Although as described earlier,lipopeptides are believed to interact with TLR2, it hasbeen suggested that an important component of theadjuvant effect of synthetic lipopeptides is their abilityto aggregate into particulate structures [132]. Moreover,the potency of lipopeptides can be enhanced by theirformulation into particulate delivery systems [133].

9. Microparticles as adjuvants

Antigen uptake by APC is enhanced by associationof antigen with polymeric microparticles, or by the useof polymers or proteins which self-assemble into parti-cles. The biodegradable and biocompatible polyesters,the polylactide-co-glycolides (PLG) are the primarycandidates for the development of microparticles asadjuvants, since they have been used in humans formany years as suture material and as controlled releasedrug delivery systems [134,135]. However, the adjuvanteffect achieved through the encapsulation of antigensinto PLG microparticles has been demonstrated onlyrelatively recently [136–139]. The adjuvant effect ofmicroparticles appears to be largely a consequence oftheir uptake into DC, macrophages and local lymphnodes following intramuscular injection. In contrast toalum, PLG microparticles are effective for the induc-tion of CTL responses in rodents against entrappedantigens [133,140,141], although attempts to induceCTL responses in primates have so far been disappoint-ing. Microparticles also appear to have significant po-tential as an adjuvant for DNA vaccines [4,142]. We


have recently described a novel approach in whichcationic microparticles with adsorbed plasmids may beused to dramatically enhance the potency of DNAvaccines [4] (Fig. 2). Importantly, the cationic mi-croparticles enhance both antibody and CTL responsesin a range of animal models, they efficiently adsorbDNA and can deliver several plasmids simultaneouslyon the same formulation, at a range of different loadinglevels [143,220]. The microparticles appear to be effec-tive as a consequence of efficient delivery of the ad-sorbed plasmids into DC [144]. Similar anionicmicroparticles can also be used for effective delivery ofadsorbed proteins and have been shown to be effectivefor CTL induction in mice [145]. In a recent study withHIV-1 vaccines, the potency of microparticles as anadjuvant was significantly enhanced by their formula-tion into MF59 emulsion [146]. A particularly attractivefeature of microparticles is their ability to control therate of release of entrapped antigens [147,148]. Con-trolled release of antigen may allow the development ofsingle dose �accines, which would result in improvedvaccine compliance, particularly in the developingworld. Although microparticles have significant poten-tial for the development of single dose vaccines, muchwork is needed to ensure the stability of antigens en-trapped in microparticles.

Polymers which self-assemble into particulates(poloxamers) [149], or soluble polymers (polyphos-phazenes) [150] may also be used as adjuvants, but thesafety and tolerability of these approaches remains tobe further evaluated. Nevertheless, the adjuvant effectof polyphosphazene polymers has recently been re-ported in a clinical trial [151].

Recombinant proteins which naturally self assembleinto particles can also be used to enhance delivery ofantigens to APC. The first recombinant protein vaccinethat was developed, based on hepatitis B surface anti-gen (HBsAg), was expressed in yeast as a particulateprotein [152]. Recombinant HBsAg is potently im-munogenic and can be used to prime CTL responses invivo [153]. HBsAg and other virus-like particles (VLP’s)can also be used as adjuvants for co-expressed proteins

[154]. For example, recombinant Ty VLP’s from Sac-charomyces cere�isiae carrying a string of up to 15 CTLepitopes from Plasmodium species have been shown toprime protective CTL responses in mice following asingle immunization [155]. In addition, Ty VLP’s havealso been shown to induce CTL activity in macaquesagainst co-expressed SIV p27 [156]. Clinical trials of TyVLP’s have shown them to be safe and immunogenic inhumans [157].

10. Alternative routes of immunization

Although most vaccines have traditionally been ad-ministered by intramuscular or subcutaneous injection,mucosal administration of vaccines offers a number ofimportant advantages; including easier administration,reduced adverse effects and the potential for frequentboosting. In addition, local immunization induces mu-cosal immunity at the sites where many pathogensinitially establish infection of hosts. Oral immunizationwould be particularly advantageous in isolated commu-nities, where access to health care professionals isdifficult. Moreover, mucosal immunization would avoidthe potential problem of infection due to the re-use ofneedles. Several orally administered vaccines are com-mercially available, which are based on live-attenuatedorganisms, including polio, Vibrio cholerae andSalmonella typhi. In addition, a wide range of ap-proaches are currently being evaluated for mucosaldelivery of vaccines [158], including many approachesinvolving non-living adjuvants and delivery systems.

The most attractive route for mucosal immunizationis oral, due to the ease and acceptability of administra-tion through this route. However, due to the presenceof low acidity in the stomach, an extensive range ofdigestive enzymes in the intestine and a protectivecoating of mucus which limits access to the mucosalepithelium, oral immunization has proven extremelydifficult with non-living antigens. However, novel deliv-ery systems and adjuvants may be used to significantlyenhance the responses following oral immunization.

11. Mucosal immunization with microparticles

In mice, oral immunization with PLG microparticleshas been shown to induce potent mucosal and systemicimmunity to entrapped antigens [152,159–162]. In addi-tion, mucosal immunization with microparticles in-duced protection against challenge with B. pertussis[163–166] Chlamydia trachomatis [167] and Salmonellatyphimurium [168]. In primates, mucosal immunizationwith inactivated SIV in microparticles induced protec-tive immunity against intravaginal challenge [169]. Alsoin primates, mucosal immunization with microparticles

Fig. 2. Serum IgG antibody titers in groups of mice (n=10) immu-nized IM with 1 or 10 �g DNA encoding HIV-1 pSS gag adsorbed tocationic microparticles or as naked DNA.


induced protection against aerosol challenge withstaphylococcal enterotoxin B [170]. Comparative stud-ies have indicated that microparticles are one of themost potent adjuvants available for mucosal delivery ofvaccines [171]. In recent studies, microparticles havealso shown some promise for the mucosal delivery ofDNA [172,173]. However, optimal responses are likelyto be achieved with microparticle formulations whichhave been modified to maintain the integrity of en-trapped DNA [174]. The ability of microparticles toperform as effective adjuvants following mucosal ad-ministration is largely a consequence of their uptakeinto the specialized mucosal associated lymphoid tissue(MALT) [175]. The potential of microparticles andother polymeric systems for mucosal delivery of vac-cines was recently reviewed [176], as was the use of abroader range of antigen delivery systems [177]. Whilemicroparticles have significant potential for mucosaldelivery of vaccines, their potency may be improved bytheir use in combination with additional adjuvants.

12. Adjuvants for mucosal immunization

The most potent mucosal adjuvants currently avail-able are the bacterial toxins from Vibrio choleraecholera toxin (CT) and Escherichia coli heat-labile en-terotoxin (LT). However, since CT and LT are tootoxic for use in humans, they have been geneticallymanipulated to reduce toxicity [178–180]. Single aminoacid substitutions in the enzymatic A subunit of LTallowed the development of mutant toxins that retainedpotent adjuvant activity, but showed negligible or dra-matically reduced toxicity [181–183]. LT mutants havebeen used by the oral route to induce protective immu-nity in mice against challenge with H. pylori [184]. Inaddition, LT mutants have been shown to be potentoral adjuvants for co-administered model antigens[185]. Oral immunization may also be achieved throughthe ingestion of transgenic plants expressing antigensand adjuvants [186,187].

Nevertheless, due to the significant challenges associ-ated with oral immunization, various alternative routesof immunization have been evaluated, including nasal,pulmonary, intravaginal and intra-rectal. Of these, in-tranasal immunization offers the most promise, bothdue to the potent responses induced by this route anddue to the easy access and simple administrationdevices which already exist. On many occasions, theability of LT mutants to induce potent antibody re-sponses following intranasal immunization has beendemonstrated [188]. In addition, intranasal delivery ofHIV-1 p24 gag with a non-toxic LT mutant inducedpotent CTL activity in mouse splenocytes. In contrast,several alternative adjuvants were ineffective for CTLinduction in the same study (Fig. 3). Several recent

Fig. 3. The induction of cytotoxic T cell responses in mouse spleno-cytes following intranasal delivery of HIV-1 p24 gag with variousadjuvants (n=5 per group). The adjuvants evaluated included PLGmicroparticles, iscoms, iscomatrix and LTK63. Adjuvants were com-pared to IN immunization with the same dose of p24 gag (25 ug)alone as a negative control and intraperitoneal immunization withvaccinia gag, as a positive control.

publications have confirmed the ability of LT mutantsto induce potent CTL responses following IN immu-nization [189–191]. Furthermore, intranasal immuniza-tion with influenza vaccine and LT mutants has beenshown to induce more potent serum immunity than IMimmunization in both small [192] and large animalmodels [193]. Importantly, this second study alsoshowed that the potency of LT mutants was not af-fected by the presence of pre-existing immunity to theadjuvant [193]. Recently, we showed that the potency ofLT mutants may be enhanced by their formulation intoa novel bioadhesive vaccine delivery system [194]. Avirosomal influenza vaccine with low dose LT wild typehas been evaluated in human clinical trials and showedpotent responses while appearing to be safe [195,196].The safety of this approach in humans, using wild typeLT intranasally, is strongly supportive of the approachusing genetically detoxified LT mutants.

Although the mechanisms of action of CT and LTremain to be fully defined, it appears that there areimportant contributions to the adjuvant effect from theB subunit binding domain, the presence of an intact Asubunit, which interacts with regulatory proteins insidecells, and also the enzymatic activity of the A1 subunit[188]. Recently, an enzymatically inactive recombinantCT mutant has been proposed to directly activate APCand T cells [197], and LT mutants may act similarly. Itis interesting to note that CTB binds to gangliosideslocalized in lipid ‘rafts’, membrane regions which arealso rich in signaling molecules, which when aggregatedcan provide co-stimulation for T cells [198]. In addition,the ability of CT to induce the activation and matura-tion of human DC has been reported [199].

Recent studies have indicated that potent mucosaladjuvants such as CT may also allow vaccination fol-lowing topical application to the skin [200] and that thisapproach may be applicable to humans [201]. In addi-


tion, epidermal immunization may be achieved usingneedle-free devices, which use helium gas to depositpowdered vaccine into the epidermis [202]. An alterna-tive approach to the development of mucosal adjuvantsinvolves the use of plant lectins [203].

13. Adjuvants for therapeutic vaccines

It seems increasingly likely that novel adjuvants mayprove sufficiently potent to allow the development oftherapeutic vaccines. Rather than prevent infection,therapeutic vaccines would be designed to eliminate orameliorate existing diseases, including: [1] chronic infec-tious diseases, e.g. those caused by HSV, HIV, HCV,HBV, HPV or H. pylori [2]; tumors, e.g. melanoma,breast or colon cancer; and [3] allergic or autoimmunedisorders, e.g. multiple sclerosis, Type I diabetes andrheumatoid arthritis. For example, a preliminary clini-cal study in subjects infected with HSV-2 showed atherapeutic benefit following vaccination with an adju-vanted recombinant vaccine [204].

The level of toxicity acceptable for an adjuvant to beused in a therapeutic situation is likely to be higherthan for a prophylactic vaccine designed to be used inhealthy individuals. Particularly if the vaccine is de-signed to treat a cancer, or a life threatening infectiousdisease. However, the acceptable safety profile for anynew vaccine/adjuvant combination needs to be estab-lished in the clinic. Many adjuvants, including MPL[205], QS21 [64] and cytokines [206] have been evalu-ated for the development of cancer vaccines.

Therapeutic vaccines may also be developed for mu-cosal administration. For example, a non-toxic LTmutant has been used to eradicate an established infec-tion with H. pylori in mice [207]. In addition, prelimi-nary studies offered some encouragement that oraladministration of antigens can result in the ameliora-tion of autoimmune diseases, including diabetes [208].

14. Future developments in vaccine adjuvants

Several recent problems have served to highlight theurgent need for the development of new and improvedvaccines. These problems have included: (1) the inabil-ity of traditional approaches to allow the successfuldevelopment of vaccines against ‘difficult’ organisms,including those that establish chronic infections, e.g.HIV and HCV; (2) the emergence of new diseases, e.g.Ebola, West Nile and nvCJD; (3) the re-emergence of‘old’ infections e.g. TB; and (4) the continuing spreadof antibiotic resistant bacteria. A likely component ofnew and improved vaccines will be more potent vaccineadjuvants. In this review, we have suggested that theadjuvants to be used in these vaccines may have to

closely mimic an infection and/or induce localized tis-sue damage to elicit protective immunity. This may beachieved through the use of particulate delivery sys-tems, which have similar dimensions to pathogens andare able to target antigens to macrophages and DC. Inaddition, it may also be necessary to deliver one ormore adjuvant active PAMP, which will more fullyactivate the innate response and may result in thedesired type of adaptive response. If this hypothesis iscorrect, it suggests that a delicate balance must bemaintained between the desired initiation of immuneresponses and avoidance of the problems potentiallyassociated with a robust response, e.g. local tissuedamage and systemic cytokine release. Many of thesenew generation vaccines will require the induction ofpotent CMI, including CTL responses. Accumulatedresearch shows that induction of CTL is difficult withproteins and may require much stronger stimulation ofthe immune system than is normally required for ahumoral response. Therefore, DNA remains an attrac-tive approach for many pathogens, but needs to bedelivered more effectively to improve its potency inhumans. In addition, live virus booster immunizationsmay also be required for optimal induction of CTL.

Targeted delivery of adjuvants and vaccines to spe-cific cell types or tissues may reduce potential toxiceffects, or help to achieve a specific desired response.Targeting may be achieved at several different levels, toinclude tissue specific delivery to local lymph nodes, cellspecific targeting to APC, or targeting to subcellularcompartments e.g. the proteasome to promote Class Ipresentation, or the nucleus for DNA vaccines. Follow-ing mucosal delivery, microparticles are ‘passively’targeted to MALT, because of their particulate nature[176]. In addition, many antigen delivery systems areeffective, largely due to their uptake into macrophagesand DC following injection, e.g. microparticles, emul-sions, liposomes and iscoms. However, ‘active’ target-ing may also be achieved through the use of ligandsdesigned to specifically interact with preferred celltypes. For example, the literature shows that lectins,have been successfully used to target antigens [209],liposomes [210] and microparticles [211] to the M cellsof MALT following mucosal delivery. In addition,lectin targeting has also been used to enhance the extentof uptake of microparticles following oral delivery[212]. An alternative targeting ligand is the mannosereceptor, which has been used to target liposomes toAPC’s [213]. Further developments in the delivery ofadjuvants may be achieved through the identification ofspecific receptors on APC, which might be extra- orintracellular. If intracellular, then a means to promoteuptake of the delivery system by the relevant cells mayalso be required for optimal efficacy. An interestingapproach to targeting APC’s has been described whichinvolves co-expression of two linked proteins, with a


targeting component and an adjuvant signal. A genefusion was created of the A1-subunit of CT and theimmunoglobulin binding domain of protein A fromStaphylococcus aureus [214–216]. The construct wasshown to be a potent adjuvant following both mucosaland systemic administration, due to it’s ability to bindto B cells and to upregulate CD86 and co-stimulatorysignals. In addition, B cell proliferation was promotedand apoptosis was prevented. This gene construct illus-trates a new strategy for the targeted delivery of adju-vant activity to a selected group of cells. However, apossible limitation of this approach is that targeting ofantigen to one specific subset of APC’s (B cells) maynot induce a sufficiently broad immune response. Al-though studies suggest that this construct has someability to induce CTL responses against a modelprotein [191]. An alternative approach to vaccinetargeting for CTL induction has also been describedusing a fusion protein with a bacterial toxin to deliverthe antigen specifically to the Class I processing path-way [217]. Targeting of adjuvants to a subset of APC’shas also been achieved through the topical applicationof vaccines, which allows antigen to gain access to theLangerhans cells in the superficial layers of the skin[176].

Future developments in adjuvants are likely to in-clude the development of more site-specific deliverysystems for both mucosal and systemic administration.In addition, the identification of specific receptors onAPC’s is likely to allow targeting of adjuvants for theoptimal induction of potent and specific immune re-sponses. However, further developments in novel adju-vants will likely be driven by a better understanding ofthe mechanism of action of currently available adju-vants and this is an area of research that requiresadditional work.

Acknowledgements

We would like to acknowledge the contributions ofour colleagues in Chiron Corporation to the ideascontained in this review: particularly Rino Rappuoli,Sergio Abrignani, Michael Houghton, John Donnellyand Gary Ott. We would also like to thank NelleCronen for help in the manuscript preparation. We aregrateful to Terry Ulrich and Charlotte Read-Kensil forthe provision of clinical data on MPL and QS21 adju-vants, respectively.

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