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Vaccine 28 (2010) 2363–2366

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

onference report

accine adjuvants: A priority for vaccine research

r t i c l e i n f o a b s t r a c t

eywords:djuvant

mmunopotentiatorelivery systemaccineoverty-related infections

The workshop on vaccine adjuvants was held in July of 2009 at the European Commission in Brus-sels, with the goal of identifying key scientific priorities as they pertain to the development of effectivevaccines against life-threatening diseases especially those associated with poverty, including HIV/AIDS,malaria and tuberculosis as well as neglected infectious diseases. On the basis of new advances in adju-vant research and related technology as well as potential challenges and roadblocks, six priorities were

evelo

eglected infectious disease identified to accelerate d

. Urgent need for novel adjuvants

Vaccines to counter several life-threatening diseases especiallyhose associated with poverty are urgently needed. The majorityf vaccine antigens currently under investigation are composed ofighly purified recombinant molecules or subunits of pathogensnd as such they lack several features of the pathogens, includ-ng the inherent immunostimulatory property, and thus ofteno not elicit strong immune responses. Despite assessments of a

arge number of adjuvants, aluminium-based mineral salts (alum)emains the only worldwide approved adjuvant for human use.lum has a good track record of safety and has been considered

he adjuvant of choice for vaccination against infections that cane prevented by antibody response, and as such has been widelynd successfully used in many licensed vaccines [1]. However, someimitations of alum have been described. Alum fails to confer ade-uate increase of antibody response to small-size peptides as wells certain vaccines such as typhoid fever and influenza vaccines.otably, alum is known to be a poor adjuvant for induction of cyto-

oxic T cell immunity and T helper 1 (Th1) responses, required toombat several life-threatening infections. Thus there is an urgenteed to develop novel adjuvants to address the development ofaccines against pathogens that have so far been refractory to theraditional vaccination strategies and to overcome the limitationsf the available licensed adjuvants.

. Mucosal adjuvants: a particular priority

Most pathogens, including HIV and Mycobacterium tuberculo-is, invade their human host or establish infection at the mucosalurfaces. However, the majority of existing vaccines are still admin-stered by parenteral injection. Mucosal vaccination, by inducing

ocal pathogen-specific immune responses, has the potential toounter mucosally transmitted pathogens at the portal of entry,hereby increasing the efficacy of vaccines [2]. Targeting mucosalnductive sites can also induce an immune response at other,emote, mucosal surfaces. Further, mucosal immunization may

264-410X/$ – see front matteroi:10.1016/j.vaccine.2009.12.084

pment of improved or novel vaccine adjuvants for human use.

offer potential advantages over the parenteral vaccination, such asincreased patient compliance due to ease of administration.

Despite these advantages very few mucosal vaccines arelicensed for human use. This is in part due to the lack of effec-tive and safe mucosal adjuvants for human use. Therefore, thedevelopment of effective and safe mucosal adjuvants representsan important priority. Bacterial toxins such as cholera toxin (CT)and heat-labile enterotoxin of Escherichia coli have been extensivelystudied as adjuvants in animal models. However, their toxicity haslimited their use for human vaccination [3]. To overcome toxicity,derivatives with no or reduced toxicity but significant adjuvantic-ity have been developed such as the non-toxic B subunit of choleratoxin (CTB) singly or in conjugation with the Toll-like receptor (TLR)9 agonist CpG [4,5], CTA1-DD molecule composed of the enzy-matically active A1 subunit of CT combined with a dimer of anIg-binding molecule from Staphylococcus aureus protein A [6], aswell as genetically detoxified mutants of LT such as LTK63 [7,8].Nasal administration of LT and CT derivatives may be unadvis-able due to their potential neuronal binding property while theiruse by the oral route is supported by strong safety records [9].Recently, TLR targeting molecules, e.g., CpG [10] and non-TLR tar-geting immunostimulators such as the natural killer T cell ligandalfa galactosylceramide [11,12] have been experimentally tested asmucosal adjuvants for vaccines against some mucosally transmit-ted infections with promising results.

The development of effective mucosal vaccines is particularlychallenging due to the unique characteristics of the mucosal envi-ronment, including the presence of the mucus, the limited antigenuptake and possible antigen degradation as well as the state ofunresponsiveness at the mucosal tissues to non-replicating anti-gens, which may otherwise lead to excessive immune responseto continuous environmental stimuli. Rational design of mucosal

adjuvants demands a better understating of the mucosal immunesystem and mechanisms governing its activation [13,14]. It iscrucial to develop standardized methods for the recovery and func-tional testing of T cells and antibodies present in the mucosal tissuesand to develop experimental systems to study T and B cell priming

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t mucosal tissues [15,16]. An alternative approach is the design ofdjuvants that imprint mucosal homing on dendritic cells followingarenteral immunization [17].

The European effort on the development of mucosal vaccinesgainst poverty-related diseases has significantly contributed todvance the field [18]. However, despite their potential, theirevelopment has so far been a low priority on the global healthgenda.

. Important areas for adjuvant research and development

.1. Understanding the mechanism of action of current adjuvants

The limited information on the mode of action of adjuvantsas led to their development on empirical basis. Recently, atten-ion has been focussed to elucidate the mechanism of action ofstablished adjuvants such as Alum and MF59 [19–22] and ofew generation of adjuvants in particular TLR agonists. Engag-

ng systems biology approaches such as OMICS technology, e.g.,ranscriptomics and proteomics in vaccine adjuvant researchas created new opportunities to comprehend their mode ofction as well as signature of adjuvanticity [19]. Such approachesay help identify novel predictors of adjuvanticity and safety,

long with pathway specific processes, which may in turn facili-ate re-engineering immune responses to target diseases. Deepernderstanding of the mechanism of action of traditional and newandidate adjuvants is thus of critical importance to inform theational design of new adjuvants with improved efficacy andafety.

.2. Harnessing the interplay between innate and adaptivemmune systems

Recent progress in understanding the function of the immuneystem and in particular innate immunity provides important infor-ation on possible new targets for adjuvants. Our innate immune

ystem can sense and respond to pathogens through a series ofattern-recognition receptor (PRRs), including TLRs, lectin-typeeceptors, and soluble cytoplasmic receptors, such as Nod-likeeceptors (NLRs) and retinoic acid inducible gene I-like receptorsRLRs). Activation of PRRs induces differential as well as overlap-ing innate immune responses. It is now becoming increasinglylear that the nature of the initial ‘danger’ signals perceived bynnate immune cells can dictate the type, quality and magnitudef the adaptive immune response. This offers unique opportunitieso custom tailor new classes of adjuvants to generate desired typesf immune response.

Current knowledge that most adjuvants act via receptors ofhe innate immune system is now driving the development of

olecular-based approaches. It has been demonstrated, for exam-le, that some classical adjuvants, including alum and saponinsctivate NLRP3 inflammasome [23,24], while complete Freund’sdjuvant contains multiple PAMPs that engage different TLRs. Lig-nds of the innate recognition systems have recently emergeds new generation of vaccine adjuvants. Thus, the TLR4 agonistonophosphoryl lipid A (MPL) has recently been approved for

uman use, and the TLR9 agonists such as CpG have reacheddvanced clinical trials [25]. Novel TLR-dependent adjuvants maymerge from the increased knowledge of TLR biology and fromhe availability of TLR functional assays that can be used to screen

ibraries of small molecules or natural products [26]. AlternativeRRs that can be exploited for the development of novel vaccinedjuvants include RLHs and NLRs such as NLRP3. Furthermore adju-ants can be designed to include �-galactosylceramide and otherutative invariant NKT cell ligands that have recently been shown

28 (2010) 2363–2366

to serve as potent mucosal adjuvants [11,12]. It may also be desir-able to combine different classes of immunostimulators as well ascytokines [27] with complementary immunopotentiating effects toachieve optimal immune activation. These would also include theuse of small inhibitory RNA to perturb immuno-regulatory factors[28].

4. Development of improved formulation and deliverytechnologies

Employment of appropriate adjuvant formulation and deliv-ery is crucial to obtain optimal vaccine effect and stability. Thechoice of formulation and delivery system depends on several fac-tors including the nature of antigen, the type of adjuvant, therequired stability, the route of immunization, and the desiredtype of immune response [29]. Other important aspects includesafety and manufacturing reproducibility. These considerationsare particularly important for mucosal vaccination where vac-cine antigens are subjected to low pH, enzymatic degradation,rapid transit and poor absorption. Liposomes and microparti-cles have the ability to encapsulate and protect antigens fromharsh conditions present in some mucosal environments, andtheir ability to target mucosal inductive sites may be improvedby the use of specific M-cell ligands. Mucoadhesive formulationsmay be critical to effective retention and delayed muco-cillaryclearance, which may facilitate penetration of vaccine com-ponents across mucosal barriers. Targeted delivery of antigenand adjuvant to antigen presenting cells is another intriguingapproach to optimize immunity and minimize systemic toxic-ity.

An attractive strategy for rational design of potent adjuvantsmay involve the combination of immunopotentiators and deliverysystems in which different components act in synergy to max-imise the adjuvant effect [29,30]. Delivery systems such as oilemulsions, adjuvant vesicles and liposomes are highly amenableto the inclusion of immunopotentiators that could potentially ren-der adjuvants more potent. One such example is the compoundAS04, a combination of alum and monophosphoryl lipid A (a TLR4agonist), which has been successfully used in the licensed humanvaccines against papillomavirus and hepatitis B virus. Other formu-lations of different immunostimulators are currently in pre-clinicaland clinical stages [30].

5. Need for standardization and cross-validation ofadjuvant research

Currently, no comprehensive overview and comparative studyof adjuvants is available. The existing data on adjuvant efficacyand safety have been generated using a variety of in vitro andin vivo protocols. This has made it difficult or even impossi-ble to compare the activity of different adjuvants. Each vaccinedevelopment project may therefore have to perform their owncomparison of a large number of adjuvants in order to selectthe most efficacious ones. Such an exercise may result in theselection of a suboptimal adjuvant, preventing the antigen fromexercising its full potential. Therefore, there is an unmet need tostandardize and cross-validate adjuvant research. Efforts should

be made to establish comprehensive and comparative guidelinesand study models for in vitro, ex vivo and animal studies as wellas human research. Such exercises necessitate the establishmentof a repository of standardized adjuvants as well as model anti-gens.

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. Safety concerns, regulatory issues and need to promoteublic-private partnership

The employment of immunostimulatory molecules as vaccinedjuvants raises theoretical safety concerns, due to the possibilityhat some might induce overt inflammatory reactions or autoim-

unity. Over the past decade many products were suggested asaccine adjuvants but rejected by the regulatory bodies due toafety concerns. The current attitude concerning the risk-benefitf vaccination puts a significant emphasis on safety as vaccines aresed in healthy people, often children, and therefore the poten-ial risk must be kept minimal. This reasonably cautious approachxercised by both pharmaceutical companies and the regulatoryodies has been the main reason for the slow pace of adjuvantevelopment and has been a major hurdle to providing open accesso safe adjuvant candidates to non-commercial partners. Adju-ants are only licensed as components of a specific vaccine andot as separate entity and the introduction of a novel adjuvantequires convincing data supporting its safety and improvement ofhe vaccine induced immunity compared to the traditionally useddjuvants [19,26]. To reach this goal, it is crucial that the regulatoryuthorities provide tools and support for the rapid advancementf safe and effective novel adjuvants from the pre-clinical to thelinical stage. Current EU regulations for the pre-clinical and clin-cal assessment of the safety of vaccines are not exhaustive [31]nd there is a further need of clear and comprehensive regulatoryuidelines that have to be constantly revised to meet the demandsf this evolving field. This is particularly critical for the developmentf novel adjuvants and on the use of novel routes of immunization.he engagement of the European regulatory authorities is crucialo help inform the regulatory framework for the development andventual approval of new adjuvants to be included in novel vaccinesor human use.

In spite of the fact that novel vaccine adjuvants are attractingew attention from vaccine researchers, it is paradoxically diffi-ult to access some of the promising new adjuvants. Therefore,any research projects on new vaccines against life-threatening

nfectious diseases, especially those carried out within the pub-ic sector use non-optimized adjuvants. Such highly pragmaticpproach may lead to otherwise promising vaccine antigenseing neglected. More efforts are needed to promote collabo-ation and flow of information between different stakeholdershrough establishment of public-private development partner-hips. This may necessitate launching an open access system forrovision of adjuvants and adjuvant information without dis-urbing the freedom-of-operation of the owner of the adjuvant32].

. Summary and final recommendations

Adjuvants form an essential and important role in the devel-pment of new vaccines to combat life-threatening diseasesspecially those associated with poverty, including HIV/AIDS,alaria and tuberculosis as well as neglected infectious diseases.evelopment of safe and effective adjuvants may also have impor-

ant implications for the improvement of the efficacy and safety ofhe existing vaccines. Increasing importance is now being placed onhe development of new, and effective use, of existing, adjuvants tonhance the immunogenicity of, and/or generate desired immuneesponse to, highly purified and well-defined antigens that can besed preferably at lower concentrations, with fewer immunizations

nd across different routes of administration. The development ofafe and effective mucosal adjuvants remains a particular prior-ty, offering the potential to induce vaccine specific responses athe mucosal portals of pathogen entry and needle free deliveryf vaccine candidates to mucosal inductive sites. New knowledge

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of molecular networks linking innate and adaptive immunity cannow be exploited in the rational design of novel adjuvants. Thesepotential important gains may be most effectively realized througha systematic open access approach to adjuvant development thatengages standardization to facilitate cross-validation of differentapproaches and that enters into effective and early dialogue withregulatory authorities to accelerate their development withoutcompromising potential safety.

Acknowledgments

We thank Natasaha Polyanskaya for organization of the work-shop. Other members of the EUROPRISE Working Group were: ElseMarie Agger, Marco Bianchi, Darrick Carter, Patrice Debre, ArnaudDidierlaurent, David Gancberg, Ennio De Gregorio, Jean Haensler,Charles Kelly, Linda Klavinskis, Thomas Lehner, Erik B. Lindblad,Anna Lonnroth, Maria Teresa De Magistris, Alessandra Martini, Far-rokh Modabber, Robbert van der Most, Eszter Nagy, Guido Poli,Manuel Romaris, Dorothea Sesardic, Silvia Vendetti, Bernard Ver-rier, Hermann Wagner, and Frank Wegmann.

Contributors: R.J.S., A.H., and D.M. organized the EUROPRISEworkshop and all authors contributed to writing the paper. Par-ticipants in the workshop contributed with formal presentationsand discussions.

Funding: Participation in the workshop was funded by theEuropean Commission. The funder provided financial support toconvene the meeting, but had no influence over the preparationof the manuscript or its submission. The authors received no addi-tional funding for the article.

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Ali M. Harandi ∗

Department of Microbiology & Immunology,Institute of Biomedicine, the Sahlgrenska Academy,

University of Gothenburg, Medicinaregatan 7A,Goteborg, Sweden

Donata MedagliniDepartment of Molecular Biology, University of

Siena, Italy

Robin J. Shattock,Department of Cellular and Molecular Medicine, St

George’s, University of London, UK

Working Group convened by EUROPRISE

∗ Corresponding author. Tel.: +46 31 7786229.E-mail address: ali.harandi@microbio.gu.se (A.M.

Harandi)

19 December 2009

25 December 2009Available online 9 January 2010