Biologicals 38 (2010) 594e601

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Biologicals
journal homepage: www.elsevier.com/locate/biologicals

Meeting report

Mode of action of adjuvants: Implications for vaccine safety and design

Batris Mastelic a, Sohail Ahmed b, William M. Egan c, Giuseppe Del Giudice b, Hana Golding d, Ian Gust e, Pieter Neels f, Steven G. Reed g, Rebecca L. Sheets h, Claire-Anne Siegrist a, Paul-Henri Lambert a, *
a

WHO-Center for Vaccinology and Neonatal Immunology, CMU, 1 rue Michel-Servet, 1211 Geneva, 4, Switzerland Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy c PharmaNet Consulting, 504 Carnegie Center, Princeton, NJ 08540, USA d Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892-0001, USA e The University of Melbourne, Department of Microbiology and Immunology, Parkville, Victoria 3010, Australia f Federal Agency for Medicines and Health Products, EMEA-CHMP, Belgium g Infectious Disease Research Institute, 1124 Columbia Street, Seattle, Washington 98104, USA h CAPT, U.S. Public Health Service, Vaccine Scientic & Regulatory Specialist, NIH/NIAID 6700B Rockledge Dr., MSC-7628, Bethesda, MD 20892-7628, USA
b

a r t i c l e i n f o
Article history: Received 23 June 2010 Accepted 27 June 2010 Keywords Vaccine Adjuvants Mode of action of adjuvants Vaccine safety

a b s t r a c t
For decades, the search for new vaccine adjuvants has been largely empirical. A series of new adjuvants and related formulations are now emerging that are acting through identied immunological mechanisms. Understanding adjuvant mechanism of action is crucial for vaccine design, since this allows for directing immune responses towards efcacious disease-specic effector mechanisms and appropriate memory. It is also of great importance to build new paradigms for assessing adjuvant safety at development stages and at regulatory level. This report reects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference, organized by the International Association for Biologicals (IABS), on the mode of action of adjuvants on 29e30 April 2010 in Bethesda, Maryland, USA, particularly focusing on how understanding adjuvants mode of action can impact on the assessment of vaccine safety and help to develop target-specic vaccines. More information on the conference output can be found on the IABS website, http://www.iabs.org/.

1. Introduction Steven G. Reed (Infectious Disease Research Institute, Seattle, USA) introduced the meeting by noting that the most widely used adjuvants, alum and oil-in-water emulsions, were introduced into clinical practice long before their mode of action was elucidated. These adjuvants were found to be effective in inducing a humoral immune response and to be non antigenic.

Abbreviations: (r)Ad, (recombinant) Adenovirus serotype; APCs, Antigen-presenting cells; CAF, Cationic liposome formulations; CRP, C reactive protein; DC, Dendritic cells; DDA, Dimethyldioctadecylammonium; GBS, Guillain Barre Syndrome; GSK, GlaxoSmithKline; HA, Hemagglutinin; HIV, Human immunodeciency virus; IFN-, Interferon-; IL-, Interleukin-; (r)LCMV, (recombinant) lymphocytic choriomeningitis virus; LPS, Lipopolysaccharide; LT, Heat-labile enterotoxin of Escherichia coli; MM6, Mono Mac 6; MoA, Mode of action; MPL, Monophosphoryl Lipid A; NK, Natural killer; PAMPs, pathogen-associated molecular patterns; PGE2, prostaglandin E2; RA, retinoic acid; TB, Tuberculosis; TDB, a,a-trehalose 6,60 dibehenate; TH, T helper; TLR(s), Toll-like receptor(s); TNF-a, tumor necrosis factor alpha; Tregs, Regulatory T cells. * Corresponding author. Fax: 41 22 3795746. E-mail address: paul.lambert@unige.ch (P.-H. Lambert). 1045-1056/$36.00 doi:10.1016/j.biologicals.2010.06.002

There are several reasons for searching for additional adjuvants, including the desire to induce (1) broader immune responses capable of covering multiple serotypes, (2) strong T-cell responses required against infections such as hepatitis C virus and human immunodeciency virus (HIV) or to form the basis of therapeutic vaccines, (3) responses which overcome immunological senescence or stimulate an immune response early in life, (4) potent mucosal immunity, (5) immune responses to poorly immunogenic antigens or (6) to allow manufacturers to reduce the dose of antigen, thus reducing cost and increasing the number of doses of vaccine that can be produced. Advocates of novel adjuvants aim at tailoring the vaccineinduced immune responses to achieve maximal efcacy, through optimization of B-cell responses (in terms of level, quality, duration and memory) and generating appropriate T-cell responses (in terms of effector functions and memory). Their expectation is that understanding which innate immune receptors are involved in the response to different classes of adjuvants, and which signals convert dendritic cells (DC) and monocytes into immunostimulatory antigen-presenting cells (APCs), will provide a scientic rationale for their use, and reduce the risk of unintended adverse reactions.

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2. Update on the adjuvant mode of action 2.1. Aluminum-containing adjuvants Mirjam Kool (Ghent University, Ghent, Belgium) discussed aluminum-containing adjuvants (typically referred to as alum) which are widely used in human vaccines, although their mode of action is not completely understood. Alum is known to stimulate humoral immunity and strong T helper (TH) 2 responses [1]. Adsorption of antigens to alum allows them to be presented in a particulate form that can be phagocytosed by APCs. Alum also forms a depot at the injection site from which the antigen is released gradually, enhancing the likelihood of capture by APCs, hence increasing antibody production (Reviewed in ref. [2]). However, this has been challenged by a report demonstrating that adsorption and retention of the antigen at the injection site is not always required for immunopotentiation [3]. The immunostimulatory property of aluminium salts has recently been shown to be DC-dependent [4], whereas the T-cell response partially depends on Nalp3 inammasome activation [5]. Nalp3 belongs to the NOD-like receptor (NLRs) family of intracellular recognition receptors, which can detect stimuli of microbial origin as well as endogenous molecules generated during cellular damage (e.g. ATP or crystals of monosodium urate) [6]. Nalp3, together with ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and caspase-1, form the inammasome, which regulates the cleavage and release of the potent pro-inammatory cytokines interleukin (IL)-1b, IL-18 and IL-33 [7]. Alum induces strong production of uric acid, thereby activating the Nalp3 inammasome and inducing the production of the pro-inammatory cytokines IL-1b and IL-18 [8]. Aluminiumcontaining adjuvants, as well as crystals of monosodium urate, induce the recruitment and differentiation of inammatory monocytes (F4/80intCD11bLy6GLy6C), which differentiate into inammatory DC [8]. These inammatory DC are potent inducers of CD4 T-cell activation and proliferation and thereby of antibody production. In contrast, when mice were administered uricase (an enzyme that rapidly breaks down uric acid), the recruitment of inammatory monocytes was abolished, along with a reduction in IL-1b production and T-cell response, indicating that, in this model, uric acid is crucial for alums effect [8]. 2.2. Toll-like receptors agonists As stressed by Dennis Klinman (National Cancer Institute at Frederick, Frederick, USA) the discovery of pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and CpG motifs, as powerful activators of the immune system through toll-like receptor (TLR) interaction, prompted a variety of TLR agonists to be used to modulate the immune system (Table 1). Immunopotentiators act on APCs, mainly DC, which express these TLRs for screening the environment for pathogens. TLR activation
Table 1 TLRs as vaccine adjuvants targets (modied from refs. [10,11]). TLR TLR1-2 TLR2-6 TLR3 TLR4 Ligands Triacyl lipopeptides (PAM3CSK4) Diacyl lipopeptides (PAM2CSK4) dsRNA (PolyI:C) LPS Monophosphoryl lipid A (MPL) Glucopyranosylphospho-lipid A (GLA) Flagellin ssRNA; Imiquimod DNA; CpG oligonucleotides

leads to an increased recruitment of innate immune cells at the infection site, along with production of pro-inammatory cytokines and chemokines, nally resulting in the induction of antigenspecic adaptive immune responses [9]. Nathalie Garon (GlaxoSmithKline (GSK) Biologicals, Rixensart, Belgium) pointed out that the way immunopotentiators are delivered can affect their immunogenicity. New formulations have been designed to increase the antigen availability at the injection site, thus facilitating antigen uptake by APCs. Monophosphoryl Lipid A (MPL), a non-toxic bacterial LPSderivative, was the rst TLR targeting adjuvant (dependent on TLR4) approved for human use and is the basis of GSKs adjuvant AS04 (MPL & alum), which is used in the licensed hepatitis B vaccine [12] and human papillomavirus vaccine [13]. AS04 stimulates the migration and activation of DC and monocytes in draining lymph nodes, leading to the induction of the NFkB pathway. It results in an early and transient local cytokine response, which is more efcient in stimulating adaptive T and B-cell responses, including a high level of memory B cells, than alum [14,15]. It should be noted that the antigen (virus-like particles) and the adjuvant need to be co-localized in lymph nodes to have a benecial adjuvant effect on relevant APCs [15]. 2.3. Saponin-based adjuvants The importance of formulation was further indicated with adjuvants involving saponins, which are derived from the bark of the quillaia tree. The starting material, Quil A, is reactogenic and can cause toxicity in man including haemolysis [16], properties which are lost when the material is mixed with cholesterol. As indicated by Debbie Drane (CSL Limited, Parkville, Victoria, Australia), while several saponin-based adjuvants are currently in advanced clinical development, their mechanism of action is still being investigated. Thus far, saponins-based adjuvants were shown to possess antigen delivery and immunomodulatory capabilities. These include induction of a balanced TH1/TH2 response with antibody production [17], as well as cytotoxic CD8 lymphocytes [18]. Saponin complexes have been shown to utilize a MyD88-dependent (TLRindependent), and IL-18 receptor signaling pathway. CSLs saponin-based adjuvant, ISCOMATRIX, induces rapid and transient cytokine production, thus leading to a transient inux of innate cells (e.g. natural killer (NK) cells, NKT, neutrophils and macrophages) to the draining lymph nodes [19]. In addition, APCs (specically migratory DC (CD205CD8) and CD8a DC) are activated to induce cross-presentation and prolonged antigen presentation in draining lymph nodes. 2.4. Emulsions Ennio De Gregorio (Research Center, Novartis Vaccines and Diagnostics, Siena, Italy) reviewed emulsion based adjuvants. The rst used adjuvant in this category is Novartis MF59, an oil-in-water emulsion containing squalene. It is a component of

Type of immune response TH1, antibody, NK cells TH1, antibody, NK cells NK cells Strong TH1, antibodies TH1, CTL, antibodies Strong TH1, CTL Strong TH1, CTL and antibodies, NK cells

TLR5 TLR7/8 TLR9

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licensed seasonal and pandemic inuenza vaccines and was shown to increase immunogenicity and breadth of the antibody response (Reviewed in ref. [20]). Recently, the mode of action of MF59 has been dissected. An analysis of the effect of different adjuvants in mouse muscle (by microarray and ow cytometric analysis) identied MF59 as a strong immune potentiator at the injection site, resulting in early leukocyte recruitment [21]. In addition, ow cytometric analysis showed that MF59 triggered rapid recruitment of CD11b blood cells into the injected muscle. Pentraxin3 (Ptx3) and JunB biomarkers were expressed early in muscles, suggesting that MF59 directly activates muscle bers causing production of immune mediators that activate local DC [21]. It is suggested that MF59 has dual adjuvant properties, combining antigen delivery with immune potentiator activity at the injection site, leading to the recruitment and activation of circulating and resident APCs. 2.5. Liposomes Peter Andersen (Statens Serum Institut, Copenhagen, Denmark) discussed the place of liposomes, particularly cationic liposomes based on dimethyldioctadecylammonium (DDA). Cationic liposome formulations (CAF) are lipid bilayer vesicles with positive surface charge. As these particles may not be sufciently immunostimulatory, they can be combined with immunostimulators, such as a,a-trehalose 6,60 -dibehenate (TDB), (a synthetic mycobacterial cordfactor analogue). TDB has a stabilizing effect on liposomes, preventing aggregation and precipitation in suspension and this formulation is known as adjuvant CAF01 [22]. The CAF01 adjuvant system has shown promising activity in a tuberculosis (TB) vaccine in an animal model [23], and can be produced on a large scale. The system is in Phase I studies and appears to be safe. Immunogenicity data are expected shortly. The experimental CAF01 adjuvanted TB vaccine promotes depot formation, delaying the release of the antigen while targeting the antigens and immunomodulator to the same activated APCs [24]. The preparation stimulates both cellular and humoral immune responses, along with efcient polyfunctional memory T cells [23]. It promotes a TH1 biased response, plus a TH17 response, probably through involvement of the C type lectin receptor, Mincle, and Syk/ Card9 signaling cascade, activating the TH17 signaling pathway [25]. The CAF backbone could also be used with other PAMPs. A recent formulation, CAF05, which includes DDA/TDB and PolyI:C has been shown to trigger the activation of TLR3 and to prevent aggregation of liposomes, whilst preventing the release of free PolyI:C and its degradation. Unlike CAF01 which elicits a predominant CD4 response, CAF05 induces CD8 as well as CD4 responses. 3. Correlating the adjuvant mode of action with vaccine design for specic disease targets Paul-Henri Lambert (WHO Collaborating Center for Vaccinology and Neonatal Immunology, Geneva, Switzerland) introduced the issue of adjuvant selection in relation to the target disease and the type of vaccine that is needed. He stressed the importance of properly balancing the level of adjuvantation to ensure the efciency of the immune response, while avoiding excessive nonspecic responses and ensuring vaccine acceptance. In fact, measuring the type and level of innate immunity triggered by recently introduced attenuated vaccines (e.g. live inuenza) may provide a benchmark for dening an acceptable level of adjuvantation. He pointed out that presently used adjuvants are generally associated with a lower level of innate immunity activation than most live attenuated vaccines. A particular attention will

have to be given to adjuvants for persisting viral infections (e.g. herpes viruses), to counteract virally coded genes with immune down-regulation activities that may limit vaccine efcacy. 3.1. Challenges for early life vaccines adjuvants Claire-Anne Siegrist (WHO Collaborating Center for Vaccinology and Neonatal Immunology, Geneva, Switzerland) reminded that while many serious enteric and respiratory infections that occur in the rst 6 months of life are potentially preventable by immunization, B-cell responses in infants are seriously diminished [26]. Novel adjuvants may have a role in enhancing B-cell activation in germinal centers and bone marrow plasma cell survival. For instance, the non-toxic mutant heat-labile enterotoxin (LT) of Escherichia coli, LTK63, given parenterally to neonatal mice, was recently shown to overcome the delayed maturation of follicular DC and the generation of germinal centers (Bjarnarson SP. et al. submitted). T-cell responses are also affected during early life, with a trend towards TH2-type responses and reduced IFN-g and CD8 responses [27]. The reduced TLR-mediated pro-inammatory responses in newborns may have benecial effects, such as reducing the risk of alloreaction and favoring tolerance to self antigens. However, adult-like TH1-type responses can be safely induced in newborn mice using some novel adjuvants such as IC31 (antimicrobial peptide plus TLR9 ligand) [28], or CAF01 [24]. It is remarkable that adjuvants that were found to be effective in early life may differ from those effective at adult age. 3.2. The potential effect of adjuvants for vaccination in the elderly Beatrix Grubeck-Loebenstein (Austrian Academy of Sciences Institute for Biomedical Aging Research, Innsbruck, Austria) noted that in man, involution of the thymus occurs with increasing age, leading to a decreased output of nave T cells. This results in a reduced response to primary vaccination, although the ability to respond to boosters is retained [29e31]. The age-associated decrease in TLR function of DC also limits vaccine responses in the elderly [32]. While memory T cells have benecial properties in old age, effector T cells tend to have pro-inammatory properties, taking place in the lymphoid organs, which may inhibit antibody production [33e35]. It would be important to nd a way to induce a danger signal in spite of age-related increased pro-inammatory processes at the injection site. In aged populations adjuvants should particularly help to stimulate memory cells in order to achieve improved booster responses. Virosomes and MF59 have been used in inuenza vaccines in the elderly, to enhance the levels of protective antibody responses [36,37]. 3.3. Selection of an adjuvant in the development of a malaria vaccine Marcelle Van Mechelen (GSK Biologicals, Rixensart, Belgium) discussed the selection of adjuvants for GSKs candidate malaria vaccine, as recombinant malaria proteins are poorly immunogenic when administered alone. The pre-erythrocytic malaria vaccine, RTS,S (comprising part of the circumsporozoite protein co-expressed with the hepatitis B surface antigen (HBsAg) as virus-like particles), was rst tested with two of GSKs combination adjuvants, AS01 and AS02 [38]. AS02, an oil-in-water preparation containing MPL and QS21, was initially selected based on the results of a challenge trial in human volunteers. In 1e4 year-old children in Mozambique, the RTS,S/ AS02 malaria vaccine demonstrated protective efcacy (varying between 26% and 49%), accompanied by high antigen-specic IgG

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levels and strong IFN-g production [39]. Subsequently, better antibody and CD4 responses and protective efcacy were observed when AS02 was replaced with AS01, in which MPL and QS21 are formulated in liposomes [40,41]. Based on these promising data, a multicenter Phase III efcacy trial is underway in seven African countries, involving up to 16,000 children aged 6 weeks and older. 3.4. Adjuvants for tuberculosis vaccines The adjuvants for tuberculosis vaccines were covered by Stefan H.E. Kaufmann (Max-Planck-Institute for Infection Biology, Department of Immunology, Berlin, Germany). Several vaccine candidates against TB are currently in phase I/II trials. These include the fusion protein containing the antigens 85B and ESAT-6 adjuvanted with IC31, the same fusion protein adjuvanted with CAF01, and the fusion of antigens 85B and TB10.4 adjuvanted with IC31. The current focus of the present vaccine formulations is to induce persistent antigen release, activation and recruitment of antigenpresenting cells, and induction of TH1-type responses. Future directions will have to include cross-priming, polarization of TH cell populations, tissue-specic and age-specic immune responses. The potential targets of future TB vaccines are multiple. They could allow the infection, but then contain it and prevent outbreaks. Alternatively vaccines could allow a short-term infection, followed by bacterial clearance. Finally, TB vaccines could totally prevent TB infection. This might be achieved through sustained levels of TH1 and TH17 memory and effector CD4 cells. Such vaccines are designed to activate strong innate immunity through the use of appropriate agonists of TLR and other receptors, leading to migration of immune cells and local inammatory and adaptive effector immune responses. 3.5. Adjuvants for HIV vaccines Gary J. Nabel (NIAID, National Institutes of Health, Vaccine Research Center, Bethesda, USA) discussed the use of replicationdefective adenoviruses as candidate HIV vaccine vectors as a way to identify which adjuvant properties should be considered for future subunit HIV vaccines. Adenoviruses are non-enveloped double-stranded DNA viruses used to increase CD4 and CD8 T-cell effector functions against virally infected cells, and are under investigation for broad applications such as HIV, Ebola, and Marburg viruses, tuberculosis, malaria, and cancer. Adenovirus serotype (Ad) 5 is a widely used vector which has been tested in human clinical trials to deliver human immunodeciency virus (HIV-1) gene products that stimulate HIV-specic immune responses. However, a major limitation of Ad5 is the fact that pre-existing immunity to Ad5 is common in humans. Therefore, the majority of individuals have neutralizing serum antibodies to Ad5, which decrease immunity by rendering it harder to break tolerance and generate a long lasting immune response. The STEP trial that tested a Merck recombinant Ad5 (rAd5) vaccine encoding HIV-1 Gag, Pol, and Nef failed to show protection [42]. There was a non-signicant trend of higher HIV infection in Ad5 seropositive vaccines (high titers), which was later explained by other co-variates (circumcision status) with no signicant difference in post-infection set-point viral loads in vaccines compared with placebos [42]. The effects of prior vector immunity are not completely understood but in some cases it can result in diminished CD4 and CD8 immunity and can reduce vaccine efcacy. To overcome preexisting immunity associated with Ad5 vaccination, diverse strategies are being tested, including increased vaccine dose and/or prime-boost approaches. A promising strategy includes the use of

replication-defective recombinant lymphocytic choriomeningitis virus (rLCMV) vectors [43]. The LCMV glycoprotein gene is replaced with the vaccine gene to create replication-defective vaccine vectors. rLCMV was >1000-fold more potent than rAd5 in mice in generating tetramer specic CD8 T-cells response [43]. An international epidemiology study of human pre-existing adenovirus neutralizing antibodies identied another serotype with a high seroprevalence in some regions, Ad26. Current efforts at the VRC are focused on simian Ad11 and Ad16 vectors which were shown to stimulate cellular (CD3, CD8) and humoral (IgG) HIV immunity at levels comparable to rAd5 [44]. The potential of Ad vectors transduction to induce pro-inammatory cytokines and chemokines was examined in mouse and human DC. In addition to DC maturation, only simian Ad11-transduced DC produced high levels of pro-inammatory cytokines (Interferon (IFN)-a, tumor necrosis factor alpha (TNF-a)) and chemokines (macrophage inammatory protein-1b (MIP-1b)) in both mouse and human DC. However, simian rAd vectors transduction of mouse DC showed less activation than human DC [44]. A large Phase IIB HIV vaccine trial with participants from Thailand, known as RV144, was recently unblinded. A primeboost protocol of two genetically engineered vaccine candidates (Canarypox expressing multiple HIV genes, with two recombinant envelope proteins) modestly reduced the rate of HIV infection, but had no effect on viral load in those who became HIV infected despite vaccination [45]. 4. First lessons from the H1N1 pandemic Albert D. Osterhaus (Erasmus University, Department of Virology, Rotterdam, The Netherlands) noted that the performance of seasonal inuenza vaccines was far from being optimal in people who traditionally need it most; the elderly and the very young, and argued for the use of adjuvanted vaccines, which induce strong and broad immune responses. He suggested that there may be a need to establish additional correlates of protection for adjuvanted inuenza vaccines, as antibody titers (as measured by haemagglutination inhibition or neutralization assays) do not appear to be fully reliable. Some data suggest that different quality antibodies are induced by adjuvanted and non-adjuvanted vaccines. Hana Golding (Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Division of Viral Products, Bethesda, USA) discussed the role played by some adjuvants in spreading the epitope recognition by antibodies and in broadening the cross-clade neutralization of inuenza viruses. Using a phage-display library approach, her group at FDA/CBER had shown that broadly neutralizing anti-H5N1 human monoclonal antibodies and sera from immune subjects that survived H5N1 infection recognized large epitopes in the hemagglutinin (HA) envelope protein, encompassing the receptor binding region in the globular head of HA termed HA1 [46]. A properly folded large peptide (HA 28-319) was the target of this recognition. Using this tool, it was shown that non-adjuvanted and alum-adjuvanted H5N1 vaccines induced antibodies, preferentially recognizing epitopes in the conserved HA2 region of HA that form the stem of the viral spikes, which do not contain many protective targets [46]. On the contrary, MF59-adjuvanted H5N1 vaccine-induced antibody epitope spreading to the HA1 region as well as preferential recognition of sites in the neuraminidase located at or next to the sialic acid binding enzymatic site. Antibodies from MF59-H5N1 vaccinated subjects showed higher reactivity and cross-reactivity with the properly folded HA1 peptide as compared to antibodies from subjects vaccinated with non-adjuvanted vaccine, despite similar virus neutralization titers measured in traditional assays [46]. This effect of MF59 was not peculiar to H5N1 vaccine, since the

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same spreading of HA1 epitope recognition was also observed with MF59-adjuvanted pandemic H1N1 vaccine. As reported by Philip Bryan (Vigilance and Risk Management of Medicines, Medicines and Healthcare products Regulatory Agency, UK), more than 5 million doses of GSKs AS03-adjuvanted H1N1 vaccine (AS03 is an oil-in-water emulsion containing a-tocopherol, an isoform of Vitamin E) have been administered since October 2009. Intensive pharmaco-vigilance was instigated to capture any adverse events, with a particular focus on Guillain Barre Syndrome (GBS) and other autoimmune and neuroinammatory conditions. About 3000 adverse events were reported, the vast majority of which were mild and predicted from previous inuenza vaccine trials. Although twenty-two recipients of adjuvanted vaccines died, no death was vaccine-related. There was no evidence of an increased incidence of GBS and no other signals of demyelinating disorders or of neuroinammatory events were reported. Although manufactured and marketed doses are known, there is a lack of information on delivered doses which are actually an estimate, leaving the denominator unclear when considering adverse event rates. However, at least 150,000 pregnant women were exposed to the vaccine without any indication of vaccine-related adverse events. In the whole EU, more than 30 million doses of vaccine were given with a similar safety prole. A panel discussion included representatives from industry, academia and regulatory authorities. It was suggested that better global disease surveillance might have given 3 additional months to prepare for the H1N1 pandemic and, the use of cell culture to produce vaccine, rather than the traditional egg production might have saved time. Thus far, data suggest that even with the same titer, different quality antibodies were observed between adjuvanted and non-adjuvanted vaccines, leaving the eld still unclear how titer or quality relates to clinical protection. While in the non-primed animals, non-adjuvanted vaccine does not perform as well as adjuvanted vaccines, in the primed human population, the difference may not be as signicant. Although there is signicant information about the safety, there is much less information about vaccine efcacy because most doses in Europe were released after the peak of the pandemic, so attack rates after uptake were not as signicant. More information about vaccine performance and efcacy probably needs to be developed from original trial cohorts, as these data are difcult to interpret after marketing and deployment of a vaccine. The introduction of a novel vaccine containing a little known adjuvant at a time of great public interest also emphasized the need for good communication. Whereas some countries had a good rollout due to cooperation of physicians and outreach to the public, others experienced signicant miscommunication resulting in mistrust and low vaccination enrollment. Although surveillance is continuing and much of the information has not been published yet, recent experience with the safety of oil-in-water-adjuvanted H1N1 vaccines is very encouraging.

5. Can understanding the adjuvant mode of action help in assessing vaccine safety and alleviating public health concerns? 5.1. Vaccine adjuvant safety: main issues Kathryn C. Zoon (National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, USA) noted that while the development of new vaccine adjuvants is a challenging process and that it was not until 70 years after alum was introduced that oil-in-water adjuvant (MF59) became a component of a vaccine licensed in Europe, and more recently AS04 (containing MPL) was licensed in the US in combination with HPV vaccine.

She drew attention to the need for special consideration to tolerability of adjuvants likely to become components of widely used vaccines and the need for careful monitoring of local and systemic reactions as well as rare events such as autoimmune diseases and neurological disorders. Clinical studies should therefore assess these concerns, both for the adjuvant and the adjuvanted vaccine products. The manufacture and quality control of a novel adjuvant are also critical. As with other vaccine components, a consistent manufacturing process that assures batch-to-batch consistency is essential and the product must be demonstrated to be pure and sterile. Hana Golding (Center for Biologics Evaluation and Research, Food and Drug Administration, Division of Viral Products, Bethesda, USA) pointed out that non-clinical safety evaluation of adjuvanted vaccines, especially toxicology and mode of action (MoA), utilizes animal models, which should be carefully selected for their ability to mimic the outcome in humans. Some species discrepancies limit the ability of animal models to predict the effect of novel adjuvants in humans. These include differences in innate immune receptor distribution between mice and humans, in cellular sources of proinammatory cytokines that contribute to systemic and local reactogenicity and pyrogenicity and differences in anatomical structure of the skin and mucous membranes. Hana Golding discussed new in vitro approaches to non-clinical studies of novel adjuvants based on human cells. A human monocytoid cell line, Mono Mac 6 (MM6), has been used to establish an in vitro assay with the potential to predict in vivo toxicity. Endotoxin was chosen as a reference standard to establish the threshold between safe and non safe levels of pro-inammatory cytokines in MM6 cell culture (measuring IL-1b, TNF-a, IL-6 and IL-8) based on the known pyrogenicity threshold of this Endotoxin Standard in rabbits. An in vitro safety threshold for cytokine production by MM6 was established using 0.5 EU/ml of endotoxin, a level shown to induce a 0.5  C increase in rabbit body temperature. The assay was used to study adjuvants with established clinical proles, using pro-inammatory cytokine secretion as the read-out, with encouraging results. For instance, the differential toxicity of Alum and MF59 vs. saponinbased adjuvants in humans, correlates nicely with their induction of pro-inammatory cytokines in MM6 cells. To conrm and complement in vitro toxicity predictions, additional tests were undertaken in rabbits to assess pyrogenicity, measure prostaglandin E2 (PGE2) up-regulation in the plasma and C reactive protein (CRP) levels in sera, using adjuvants known to activate innate immune receptors (e.g. TLRs, NODs). These studies demonstrated a correlation between the type and dose of TLR agonists that induced increased levels of pro-inammatory cytokines in vitro and pyrogenic responses in vivo. Plasma PGE2 levels were increased before temperature elevation, while CRP elevations occurred only at 24 h. An in vitro assay for PGE2 production following TLR ligand stimulation of Phorbol Myristate Acetate (PMA) treated U937 cells, is under development. If successful, this assay is a potential model of the response of tissue macrophages such as liver Kupffer cells. These new assays could be helpful during the non-clinical screening of novel adjuvants, by excluding compounds with potential in vivo toxicity, providing guidance on dose-range and helping to rank order chemical modications designed to optimize activity and minimize toxicity. 5.2. Considering the essential steps for the early assessment of adjuvanted vaccine safety, point of view from industry Sohail Ahmed (Novartis Vaccines and Diagnostics, Siena, Italy) presented a review prepared with colleagues from GSK,

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Sano-Pasteur and Dynavax regarding some general principles that may be considered to evaluate vaccine safety. In order to accelerate development of novel vaccine adjuvants for human use, it is important to understand their MoA and their manufacturability and to evaluate their safety in non-clinical studies and in humans. He noted the growing need of collecting data in a manner that would facilitate meaningful comparison of adjuvanted products, the importance of establishing normal ranges and what constitutes out of range values, and the need to standardize reporting of serious adverse events by physicians during clinical trials. He stressed that the long-term safety of novel adjuvants can only be reliably established by well conducted epidemiological studies of licensed products containing the adjuvant. To assess the impact of an adjuvanted product, a large data base of immune proles should be established in healthy adults and ideally, in populations whose immune system may be compromised by conditions of pregnancy, transplantation, HIV infection, autoimmune disease, or age. Such data may assist in identifying potential biomarkers (cytokines, proinammatory mediators, molecular proling) that predict the onset or exacerbation of immune disorders and evaluating any potential link between the product and short- or long-term adverse events. Due to the limitations on the size of clinical trials conducted prior to licensure of a new vaccine, post-marketing and follow-up studies are essential in identifying rare adverse events, even though it may be difcult to prove a causal relationship. Indeed, adverse events accumulate over time in large cohorts and it may be difcult to establish a causal relationship to vaccination. Assessment of the data obtained during and following vaccine trials should involve experts who are independent of the trials sponsors. Finally, it would be important to determine whether preclinical animal models of human autoimmune disease induction can be developed to better evaluate vaccine/adjuvant associated risks. 5.3. Adjuvants and autoimmune diseases Jean-Franois Bach (INSERM, Hpital Necker, Paris, France) reviewed the relative risk of autoimmune diseases. Exacerbations of autoimmune diseases such as multiple sclerosis (MS) were frequently reported following viral infections in association with an expansion of auto-reactive T cells. The question of whether adjuvanted vaccines could act in the same manner was raised. This was not conrmed by multiple clinical studies. One strategy, presented by Robert S. Fujinami (University of Utah School of Medicine, Department of Pathology, Utah, USA), is to assess the risk of autoimmune responses using animal models of autoimmunity, such as experimental autoimmune encephalomyelitis (EAE), which is used as a model of MS. However given the limitations of those models, any signal should be interpreted cautiously. 5.4. Special issues with mucosal adjuvants Yasmine Belkaid (NIAID, National Institutes of Health, Laboratory of Parasitic Diseases, Bethesda, USA) discussed the importance of gut commensal microora in controlling host immune homeostasis. Gut immunity depends on the interaction between host cells, commensals and pathogens, which can have an impact on systemic responses, including allergy and autoimmunity. Together with vitamin A metabolite, retinoic acid (RA), regulatory T cells (Tregs) were dened as key players in regulating gut homeostasis, at healthy steady state or during infection. Using germ free animal models, the contribution of commensals and Tregs to the immune system was demonstrated. Indeed, during infections, removal or modication of the gut ora was associated with signicant modications in the phenotype of the host responses [47]. Therefore, some microorganisms may interfere with Tregs present in the

gut lymphoid tissues in order to guarantee their own survival. RA was shown to induce Tregs generation, while inhibiting the generation of TH17 cells, via mucosal CD103 DCs subset. In addition, using mice decient for RA receptor a (RARa), RARa was identied as essential for TH1 and TH17 cell priming. Thus, it is hypothesized that signals derived from the gut ora are acting as adjuvants of immune responses for priming intestinal responses against oral pathogens via modulation of Tregs and T effector cells responses. Myron M. Levine (University of Maryland School of Medicine, Center for Vaccine Development, Baltimore, USA) discussed the need for novel mucosal adjuvants, so that vaccines can be delivered mucosally, avoiding childrens fear of needles and inadvertent transmission of blood borne infections. Since mucosal surfaces are constantly exposed to exogenous (food, inhaled pollens) and endogenous (normal ora) antigens, to which they are tolerant, it is important to demonstrate that mucosal adjuvants do not inadvertently induce immune responses to these bystander antigens. Cholera toxin (CT) and E. coli heat-labile enterotoxin (LT) are powerful mucosal adjuvants that enhance immune responses to coadministered vaccines, although both can cause clinically relevant diarrhea when administered alone. To allow broader use, non-toxic mutants have been engineered and administered intranasally. Although devices have been designed to avoid delivery of aerosol particles to the lungs, other safety concerns remain; especially the possibility that LT can utilize nerve bers in the nose to access the olfactory bulb of the brain. An inactivated inuenza vaccine adjuvanted with LT and administered intranasally, was licensed in Switzerland but rapidly withdrawn from the market after postmarketing surveillance identied an increased risk of Bells palsy in recipients. Even though the development of intranasally administered adjuvanted vaccines stopped, a live attenuated inuenza which is administered by this route has been licensed, and an aerosolized live attenuated measles vaccine is under evaluation. 5.5. Overall discussion on adjuvant safety A panel discussion included representatives from industry, academia and regulatory authorities. It was suggested that concerted efforts were needed to move the eld forward. Shortterm priorities include harmonized denition, identication and evaluation of serious adverse events across clinical trials. In-depth study of the mode of action of novel adjuvants may help in assessing risk including the possibility of triggering or exacerbating an autoimmune event. Mid-term priorities include assessment of background rates of incidence and prevalence of autoimmune diseases depending on age, race and geography to assist in interpreting clinical data. Identifying and validating new biomarkers of adjuvant-induced toxicities in both animal models and humans may improve safety evaluations in preclinical studies and vaccine trials in humans. A long-term priority is to determine whether animal models can be developed that reliably predict vaccine/ adjuvant associated risks in humans. The panel noted that because of the complexity of the immune system and the fact that many adjuvants have complex components with mixed effects on different cell types in vivo, complete understanding of their MoA might not be achievable prior to licensing, and must await future technological advances and scientic knowledge. 6. Concluding remarks Paul-Henri Lambert and William M. Egan (PharmaNet Consulting, Princeton, USA) stressed again that licensing a vaccine containing a novel adjuvant requires convincing data supporting the products safety and efcacy. Whereas a detailed understanding of

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the mode of action of the adjuvant is highly desirable, it has not prevented the licensing of new products when the risk benet ratio is regarded as acceptable. Since new technologies such as transcriptomics and proteomics are now available, there are prospects of unraveling the adjuvant MoA during non-clinical studies. Although there is growing knowledge based on understanding that innate immune receptors are involved in adjuvant mechanisms, understanding their MoA would help in generating hypotheses regarding efcacy and toxicity and address safety considerations. As each sponsor of a novel adjuvant establishes their own evaluation criteria it would be useful to establish a set of basic immune functions that should be monitored before and during clinical studies and to establish the range of responses to current vaccines, which could be used for comparative purposes. Although this is an ideal outcome for regulatory authorities, it is impractical for pharmaceutical companies to delay their programs until these questions have been addressed. In preclinical studies, animal models are useful in identifying the optimal antigen/adjuvant ratio, measuring changes in hematological and immunological parameters, and toxicology evaluation and providing guidance for Phase I clinical studies. Valuable additional data may be obtained by the use of human cell lines in vitro to detect potentially pyrogenic products. Classifying novel adjuvants based on the type of immune response they induce may be helpful in focusing on which issues need to be addressed in depth and to guide the clinical strategy. A more comprehensive program for collection, storage and archiving of clinical data and samples should be established. In particular a longer follow-up of subjects would be helpful so that if adverse events emerged at later time points, there would be samples available to go back to. Many fundamental questions on the mode of action of adjuvants remain unanswered and will only be addressed through basic research. Academic research could improve ow cytometry (multicolor) assays and enable deeper investigation into cellular and humoral responses. The use of more sophisticated analyses, such as microarrays and extensive T-cells proling, may provide new insights while validated assays for measuring cell mediated immunity will enable meaningful comparisons between different data sets. In conclusion, there is a real need for novel adjuvants in order to increase and direct the immune response. These would help not only in the development of vaccines against diseases such as malaria, tuberculosis and pandemic inuenza, but in developing vaccines in which the antigens of interest are poorly immunogenic. Choosing the right adjuvant for a particular vaccine depends on a number of considerations including knowledge of disease pathogenesis and correlates of protection (if known), vaccine goals, the antigens involved, the recipient population, and an understanding of the MoA of the adjuvant. The challenge, for all involved in the development of products utilizing novel adjuvants, is to address the gaps in our knowledge to dene the criteria for acceptability and dene the pathway to licensure. Increased efforts in understanding the MoA will form a critical part of this process. Acknowledgements We thank Betty Dodet and Sverine Seemann for the excellence of the meeting organization. This workshop was made possible by unrestricted educational grants from Brenntag, CSL, European Adjuvant Advisory Committee (EAAC), GlaxoSmithKline, Merck, Novartis Vaccines, Sano-Pasteur and Sano Pasteur MSD.

Appendix A. List of participants Sohail Ahmed, Novartis Vaccines and Diagnostics, Siena, Italy; Peter Andersen, Statens Serum Institut, Copenhagen, Denmark; Jean-Franois Bach, INSERM, Hpital Necker, Paris, France; Norman W Baylor, CBER-FDA, Ofce of Vaccines Research and Review, Rockville, USA; Yasmine Belkaid, NIAID, National Institutes of Health, Laboratory of Parasitic Diseases, Bethesda, USA; Philip Bryan, Vigilance and Risk Management of Medicines, Medicines and Healthcare products Regulatory Agency, UK; Robert L. Coffman, Dynavax Technologies Corp., Berkeley, USA; Ennio De Gregorio (Research Center, Novartis Vaccines and Diagnostics, Siena, Italy; Giuseppe Del Giudice, Novartis Vaccines and Diagnostics, Siena, Italy; Debbie Drane, CSL Limited, Parkville, Victoria, Australia; Kathryn M. Edwards, Division of Pediatric Clinical Research, Vanderbilt Kennedy Center, Vanderbilt University, Nashville, USA; William M. Egan, PharmaNet Consulting, Princeton, USA; Martin Friede, WHO, Initiative for Vaccine Research, Geneva, Switzerland; Robert S. Fujinami, University of Utah School of Medicine, Department of Pathology, Utah, USA; Nathalie Garon, GSK Biologicals, Rixensart, Belgium; Hana Golding, Center for Biologics Evaluation and Research, Food and Drug Administration, Division of Viral Products, Bethesda, USA; Beatrix Grubeck-Loebenstein, Austrian Academy of Sciences Institute for Biomedical Aging Research, Innsbruck, Austria; Marion Gruber, CBER, FDA, OVRR, Bethesda, USA; Ian Gust, The University of Melbourne, Department of Microbiology and Immunology, Victoria, Australia; Stefan H.E. Kaufmann, Max-Planck-Institute for Infection Biology, Department of Immunology, Berlin, Germany; Dennis Klinman, National Cancer Institute at Frederick, Frederick, USA; Mirjam Kool, Ghent University, Ghent, Belgium; Paul-Henri Lambert, CMUCenter of Vaccinology, Geneva, Switzerland; Myron M. Levine, University of Maryland School of Medicine, Center for Vaccine Development, Baltimore, USA; Gary J. Nabel, NIAID, National Institutes of Health, Vaccine Research Center, Bethesda, USA; Pieter Neels, Federal Agency for Medicines and Health Products, EMEACHMP, Belgium; Albert D. Osterhaus, Erasmus University, Department of Virology, Rotterdam, The Netherlands; Steven G. Reed, Infectious Disease Research Institute, Seattle, USA; Rebecca Sheets, USPHS, NIH/NIAID, Bethesda, USA; Claire-Anne Siegrist, WHO, Center for Vaccinology and Neonatal Immunology, Geneva, Switzerland; Elizabeth M. Sutkowski, CBER, FDA, OVRR, Bethesda, USA; Emanuelle Trannoy, Sano Pasteur, France; Marcelle Van Mechelen, GSK Biologicals, Rixensart, Belgium; Kathryn C. Zoon, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, USA. References
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