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Biologicals xxx (2012) 1e10

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Biologicals

journal homepage: www.elsevier .com/locate/bio logicals

Meeting report

Potential use of inflammation and early immunological event biomarkers inassessing vaccine safety

Béatris Mastelic a, David J.M. Lewis b, Hana Golding c, Ian Gust d, Rebecca Sheets e, Paul-Henri Lambert a,*aWHO Center for Vaccinology and Neonatal Immunology, University of Geneva, CMU, 1 rue Michel-Servet, 1211 Geneva 4, SwitzerlandbClinical Research Centre, University of Surrey, Egerton Road, Guildford GU2 7XP, UKcCenter for Biologics Evaluation and Research, Food and Drug Administration, Division of Viral Products, Bethesda, MD, USAd The University of Melbourne, Department of Microbiology and Immunology, Parkville, Victoria 3010, AustraliaeCAPT, U.S. Public Health Service, Vaccine Scientific & Regulatory Specialist, NIH/NIAID, 6700B Rockledge Dr., MSC-7628, Bethesda, MD 20892-7628, USA

a r t i c l e i n f o

Article history:Received 24 July 2012Received in revised form2 October 2012Accepted 16 October 2012

Keywords:VaccineBiomarkersVaccine safety

Abbreviations: Adj, adjuvant; AE, adverse events; AAS, adjuvant system; BET, bacterial endotoxin testdraining lymph nodes; DTaP, diphtheria, tetanus, anEAE, experimental autoimmune encephalomyelitis; Hheat shock proteins; LAIV, live attenuated influenza valysate test; LPS, lipopolysaccharide; MAT, monocyte acIMmune In vitro Construct; MM6, Mono Mac 6; MPLNLRPs, nucleotide-binding oligomerization domain, ldomain containing proteins; OVA, ovalbumin; PBMnuclear cell; PPD, purified protein derivative of tubercTH, T helper; Thmem, T helper memory; TIV, trivalentlike receptor; TT, tetanus toxoid.* Corresponding author. Fax: þ41 22 3795746.

E-mail address: paul.lambert@unige.ch (P.-H. Lam

1045-1056/$36.00http://dx.doi.org/10.1016/j.biologicals.2012.10.005

Please cite this article in press as: Mastelicvaccine safety, Biologicals (2012), http://dx.

a b s t r a c t

Highly effective vaccines have traditionally been designed in a rather empirical way, often withincomplete understanding of their mode of action. Full assessment of efficacy and reactogenicity takestime and, as a result, vaccine introduction to the market is usually slow and expensive. In addition, in rarecases, unacceptable reactogenicity may only become apparent after years of development or evenwidespread use. However, recent advances in cell biology and immunology offer a range of new tech-nologies and systems for identifying biological responses or “biomarkers” that could possibly be used toevaluate and predict efficacy and safety during vaccine development and post-marketing surveillance.

This report reflects the conclusions of a group of scientists from academia, regulatory agencies andindustry who attended a conference on the potential use of biomarkers to assess vaccine safety whichwas held in Baltimore, Maryland, USA, from 10 to 11 May 2012 and organized by the InternationalAssociation for Biologicals (IABS). The conference focused particularly on determining which biomarkersmight relate to vaccine efficacy and reactogenicity and whether our knowledge base was sufficientlyrobust at this time for the data to be used for decision-making. More information on the conferenceoutput can be found on the IABS website, http://www.iabs.org/.

1. Introduction

Vaccines are one of the most cost effective health interventions;the development process is both time consuming and costly. Strictregulatory oversight is driven by the fact that vaccines are generallyadministered to healthy individuals and that even rare side effects

PCs, antigen presenting cells;; DC, dendritic cells; dLNs,d acellular pertussis vaccine;DM, house dust mite; Hsp,

ccine; LAL, limilus amebocytetivation test; MIMIC, ModularA, Monophosphoryl Lipid A;eucine rich repeat and pyrinC, peripheral blood mono-ulin; RPT, rabbit pyrogen test;inactivated vaccine; TLR, Toll-

bert).

B, et al., Potential use of infldoi.org/10.1016/j.biologicals.2

can create significant problems if the vaccine is administered totens or hundreds of millions of people. At present, each new vaccineis evaluated for safety, and efficacy in clinical trials performed inhealthy individuals. A range of formulations and doses may betested to define the optimal level of immunogenicity and accept-able reactogenicity. As this is often an iterative process, there isa desire to improve preclinical safety predictions by defining bio-logical correlates of protection or biological markers (biomarkers),which could predict vaccine efficacy or safety earlier in develop-ment. This desire is driven both by economic and opportunitycost considerations, and by public health considerations tominimize the number of people potentially put at risk in clinicaltesting.

As reviewed in [1], a biomarker is defined as a characteristicthat is objectively measured at a single time point and evaluatedas an indicator of a physiological or pathological process, orpharmacological response(s), to a therapeutic intervention [2].In vaccinology, the search for biomarkers of vaccine efficacyfocus on identifying correlates of vaccine-induced protectiveimmunity, or of immune responses that accompany protection

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but are not necessarily causally related [3]. Such markers wouldfacilitate screening and rank-ordering the vaccine candidates inthe early stages of preclinical development. Likewise, biomarkersfor safety could potentially predict and prevent severe adverseevents, including rare events that might take years to identify inhuman populations, such as an increased risk for autoimmunediseases. Increased inflammatory reactions and systemic events,including fever, could be detected early in clinical or preclinicalevaluation. Numerous studies are underway, using multiplexedassay technologies, proteomics, transcriptomics, and metab-olomics, to compare gene expression profiles among differentpopulations following immunization, in an attempt to identifycorrelates of vaccine performance, other than the empiricalcorrelation of antibody levels with vaccine efficacy, which maynot always represent the true mechanism of protection. Usingthese concepts, it is hoped to be able to predict the performanceof vaccines in different groups such as healthy adults, children,pregnant women, the elderly, and individuals with pre-existingdiseases.

2. Vaccine-induced inflammation

2.1. Inflammation in human health: description of theinflammasomes and their activation pathway

The “inflammasome” was reviewed by Alessandra Mortellaro(Singapore Immunology Network (SIgN); Agency for Science,Technology and Research A*STAR, Singapore). Inflammasomesare multi-protein cytoplasmic complexes that facilitate activa-tion of pro-inflammatory caspases and mediate initiation ofinflammatory processes via lL-1b and IL-18, which are potentmodulators of both innate and adaptive immunity. Inflamma-some activation can be triggered by exposure to whole livebacteria, fungi or viruses, pathogen components and toxins [4].The Nucleotide-binding oligomerization domain, Leucine richRepeat and Pyrin domain (NLRP) containing 3 (NLRP3) inflam-masome contributes to dendritic cell and macrophage detectionof endogenous danger molecules, and can signal the presence ofATP, crystals of monosodium urate or calcium pyrophosphate,and other large particulates of non-microbial origin such assilica, asbestos and alum [5]. Inflammasomes may sense viralDNA, and single- or double-stranded RNA [6]. Over-activation ofinflammasomes may result in harmful inflammatory states [7],and specific polymorphisms in NLRP genes could contribute tothe susceptibility to more common multifactorial diseases suchas autoimmune disorders, arthritic diseases, diabetes, andcancer. Eicke Latz (Institute of Innate Immunity, University ofBonn, Germany and Division of Infectious Diseases, UMass MedSchool, USA) described how crystals such as alum, uric acid [8]and amyloid [9], lipids and aggregates could activate theinflammasome via lysosomal swelling and rupture. To initiatemaximal effect, crystals should be smaller than 1 mm, numerous,and in the correct molar ratio. In the discussion, it was notedthat it remains controversial as to whether aluminum saltadjuvants induce higher antibody levels after immunization byinflammasome activation, and although adjuvants may inducein vitro inflammasome formation, the link between this and thein vivo immunological outcome remains unclear [10]. NLRP3inflammasome played no role in the production of the T helper(TH) 1-associated IgG2a, IgG2b and IgG2c upon immunizationwith aluminum salt. However, influence of NLRP3 on TH2-related isotypes is still debated. Indeed, in some reportsIgG1 antibodies seemed either essential [11,12] or dispensable[13e15], whereas IgE antibodies were shown to be dependenton NLRP3 [12,14].

Please cite this article in press as: Mastelic B, et al., Potential use of inflvaccine safety, Biologicals (2012), http://dx.doi.org/10.1016/j.biologicals.2

2.2. Current assessment of vaccine reactogenicity

Jeffrey Roberts (FDA/CBER/OVRR, Bethesda, MD, USA) reviewedthe present preclinical and clinical scoring methods for assessinglocal and systemic vaccine reactogenicity. Vaccine reactogenicityrefers to a subset of adverse events that describe and are charac-terized by the inflammatory response to vaccination. This is onecomponent of the overall safety profile of a vaccine. An acceptablelevel of reactogenicity for a given vaccine depends on many factorsbut is generally very low, because vaccines are typically intendedfor healthy populations in order to prevent disease. The ability toassess and to predict vaccine reactogenicity in preclinical systems isfairly limited; although some biomarkers, such as C-reactiveprotein (CRP), show promise. Therefore, careful systematic assess-ment of reactogenicity -both local (e.g., pain, tenderness, erythema,induration) and systemic (e.g., fever, nausea/vomiting, diarrhea,headache, fatigue, myalgia) responses - in clinical trials remainsa critical component of vaccine development programs. The Centerfor Biologics Evaluation and Research (CBER) encourages the useof the toxicity grading scale guidance for healthy adults, whichis available online (http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm074775.htm). Care must be taken when adapting the gradingscale for specific populations (e.g., age, pregnancy, subjects with co-morbidities); this is an important consideration when conductingvaccine clinical trials in these increasingly key target groups.

Examples like the whole cell pertussis and smallpox vaccineshighlight the issues surrounding potentially serious adverse reac-tions associated with vaccine reactogenicity. Further research (on,for example, clinical and laboratory biomarkers) could be useful tobetter predict the overall level of reactogenicity and to explore theeffects of reactogenicity in different populations (stratified by age,gender, etc). Meanwhile, CBER encourages thorough and preciseassessment of reactogenicity, with uniform criteria in human clin-ical trials and systematic collection of a specific set of solicitedadverse events, in order to improve comparison of safety dataamong groups in the same study as well as between differentstudies.

2.3. Alternative in vitro methods for monitoring vaccinereactogenicity in preclinical studies

As stressed by Ingo Spreitzer (Paul-Ehrlich-Institut, Langen,Germany), alternative in vitro methods, such as the monocyteactivation test (MAT), have been validated [16,17] and are nowavailable to complement existing evaluations of vaccine safety. Thisis in keepingwith the policy to replace, reduce, and refine the use ofanimals in product safety testing or the so-called 3Rs. Initially, theMAT was developed as part of a 3Rs (Replacement, Refinement andReduction) strategy to substitute the classical Rabbit Pyrogen Test(RPT) [18], where temperature increase is monitored in rabbits for3 h after i.v. application of the test substance. The MAT is based onthe in vitro activation of human monocytoid cells by pyrogens (alsovalidated with cryopreserved human whole blood), which leads tothe release of pro-inflammatory cytokines with pyrogenic proper-ties (IL-1b, IL-6 and TNF-a) that are either detected by ELISA, flowcytometry or bead-array [19e21]. Although all the MAT tests thatpassed the validation were comparable to, or better, (in terms ofspecificity and sensitivity) than the RPT, the MAT cannot beconsidered as an exact replacement of the RPT as it measures thepropensity of a material to release pro-inflammatory cytokines,rather than induce fever per se. However, it has been added to theofficial pharmacopoeia as an alternative test to detect pyrogeniccapacity (European Pharmacopeia in 2010), together with RPT andthe bacterial endotoxin test [22], often referred to as the Limulus

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Amebocyte Lysate test [23], which specifically quantifies endotoxincontent, one component that can induce fever in productrecipients.

Vaccines are currently tested for endotoxins by the LAL test,which has proven to be highly sensitive and specific for endotoxin(Lipopolysaccharide (LPS)) detection, but does not provide infor-mation on pyrogenicity and potential immune activation by otherproduct components [24]. As the new generation of adjuvantsincludes some less toxic derivatives of LPS, such as Mono-phosphoryl Lipid A (MPLA), LAL becomes limited for further pyro-genicity testing of products with several immune-relevantcomponents (including other strong activators of innate receptors).Although modifications of the basic MAT approach should beconsidered for a product-specific approach, depending on thediverse immunization strategies and novel vaccines and adjuvants,it was suggested running MAT in parallel to the LAL test for theroutine lot release safety estimation of vaccine products [25].Indeed, MAT is a reliable assay for pyrogenicity testing and closelyresembles the human physiological response. Moreover, it couldgive additional information into the mechanisms of pyrogenicityand acute pro-inflammatory reactions in patients, which cannot bereached with the actual LAL test.

Hana Golding (Center for Biologics Evaluation and Research,Food and Drug Administration, Division of Viral Products, Bethesda,USA) described the new in vitro approach based on a humanmonocytoid cell line, Mono Mac 6 (MM6), which has the potentialto predict in vivo adjuvant toxicity [26]. Endotoxin was chosen asa reference standard to establish the threshold between safe andnon-safe levels of pro-inflammatory cytokines in MM6 cell culture(measuring IL-1b, TNF-a, IL-6 and IL-8), based on the knownpyrogenicity threshold of this endotoxin standard in rabbits. Anin vitro safety threshold for cytokine production by MM6 wasestablished using 0.5 EU/ml of endotoxin, a level shown to inducea 0.5 �C increase in rabbit body temperature. The assay was used tostudy TLR agonists (FSL-1, Pam3CSK4, flagellin, and R848) oradjuvants with established clinical profiles (Alum, MF59, Poly I:C,or MPL), using pro-inflammatory cytokine secretion as read-out.For instance, TLR agonists (Pam3CSK4, FSL-1, and flagellin), butnot Alum, MF59, Poly I:C, or MPL adjuvants, induced pro-inflammatory cytokines in MM6 cells above the safety threshold[26].

In addition, a correlation was found between the type and doseof TLR agonists that induced increased levels of pro-inflammatorycytokines in vitro, and pyrogenic responses in vivo [26]. In fact,pyrogenicity was further assessed in rabbits by measuring prosta-glandin E2 (PGE2) up-regulation in the plasma and showedincreased PGE2 levels before temperature elevation (with variousTLR agonists but not with alum, MF59, MPL, or Poly I:C adjuvants),while C-reactive protein (CRP) up-regulation in sera occurred onlyat 24 h [26]. As good correlations were demonstrated between thein vitro assays and rabbit studies, it has been suggested that thetesting of multiple pro-inflammatory cytokines and PGE2 release,by pro-monocytic cells such as MM6 during the non-clinicalscreening of novel adjuvants, may help in down-selection of newadjuvant candidates with less potential in vivo toxicity, in providingguidance on dose-range, and subsequently giving insight into themode of action of the tested products. As Dr. Spreitzer highlighted,each of these novel assays measure subtly different aspects ofinflammation and pyrogenicity, and a challenge for the future willbe to integrate these new parameters into our applied under-standing of in vitro assays for pyrogenicity in humans in a usefulway that does not over-complicate the evaluation. A practicalexample of the use of MAT in the retrospective investigation ofreactogenicity of an influenza vaccinewas given in the presentationof Eugene Maraskovsky (section 3.4).

Please cite this article in press as: Mastelic B, et al., Potential use of inflvaccine safety, Biologicals (2012), http://dx.doi.org/10.1016/j.biologicals.2

2.4. Whole blood fingerprint as a vaccine safety classifier in non-human primates

I-Ming Wang (Merck Research Laboratories, West Point, PA,USA) presented a novel genome-wide approach to evaluate vaccinesafety and efficacy by immunoprofiling licensed (Adacel, Menactra,Havrix, Prevnar and RabAvert) and experimental vaccines (V512 isan influenza vaccine based on the ectodomain of the M2 proteinconjugated to Outer Membrane Vesicles of Neisseria meningitidisserotype B11; MRKAd5-gag is an HIV vaccine based on a replicationdeficient adenovirus type 5 expressing HIV gag.) using the blood ofimmunized non-human primates. The aim of this approach is tolink gene expression profiles in response to vaccination in the bloodof non-human primates, with known reactogenicity to theseproducts in man. Briefly, whole blood mRNA profiling for eachvaccine was generate from rhesus monkeys at pre-vaccination,along with early (4e24 h) and late (1e2 weeks post-vaccination)post-vaccination time points. The significance of gene expressionmodulation by individual vaccines was assessed by one-wayANOVA analysis and used to generate a heat map for the derivedgene signatures. Overall, each vaccine induced a unique blood geneexpression pattern across time points, although some commonfeatures could be detected. Using a gene-module analysis approach[27], it was shown that all vaccines had time-dependent kinetics forgene up- or down-regulation [22]. For instance, at early time pointsall vaccines up-regulated genes associated with innate immunity,cytokine production and responses to virus infection, includingIFN-inducible genes, whereas, after one week, gene clusters linkedto T cell receptor signaling, cell cycle progression, and response tostress were up-regulated [22]. Notably, one of the modulesenriched in IFN-inducible genes correlated to systemic adverseevents (AE) and could thus be used to predict potential reac-togenicity [22]. Also, these findings are consistent with a previoustrial conductedwith healthy adults that assessed local and systemicAE after receiving smallpox vaccine. Following genotyping, twoSingle Nucleotide Polymorphisms (SNPs) were found in theinterferon-regulatory-factor-1 (IRF1) gene, a key transcriptionfactor regulating IFN response, and associated with the risk ofAE [28].

Therefore, this approach may provide in a not too distant futurea way to predict potential reactogenicity of novel vaccines/adju-vants and their mode of action in a preclinical model. Severalparticipants pointed-out the drawbacks of introducing primatemodels into the routine evaluation of vaccines, but it was noted thatMerck was opting to use this model more frequently on the basisthat it provided closer approximation to clinical outcomes.

3. Monitoring of vaccine-induced innate immunity

New in vitro models and biomic platforms (genomics, tran-scriptomics, proteomics and metabolomics) and the greaterappreciation of the crucial role of the innate immune response intuning immune responses offer new possibilities to further definethe early steps of vaccine responses and to delineate the mode ofaction of vaccines and adjuvants. New multiplexed profiling assaysallow researchers to identify early gene expression, identify cyto-kine or chemokine patterns that could correlate with safety andefficacy of the vaccine product, and provide valuable informationfor predicting the subsequent vaccine-specific immune response orindividual sensitivity to adverse events in clinical trials.

3.1. In vitro models of the human immune system

William Warren (VaxDesign Campus, sanofi pasteur, Orlando,FL, USA), proposed that the in vitromodel termedMIMIC� (Modular

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IMmune In vitro Construct) offers a further opportunity to reduceanimal use in human vaccine safety assessment [29]. MIMIC� useshuman circulating peripheral blood mononuclear cells (PBMC) toreplicate ex vivo the donor’s own innate and adaptive immuneresponses in the peripheral tissues of the body. This culturestrategy attempts to reproduce in vivo conditions, and is thought tobe more sensitive and accurate than standard PBMC assays. Oneapplication of the MIMIC� system is to model transendothelialmigration of monocytes and their autonomous differentiation intoantigen presenting cells (APCs), for sensitization of naïve CD4þ Tcells and the induction of antigen-specific primary human T helperand memory cell responses, as well as antigen-specific antibodyresponses.

Several commercially available vaccines and vaccine candidateshave already been tested using this platform, including trivalentinactivated influenza (Fluviron�, Flumist�, Fluzone�) [30], menin-gococcal B and yellow fever vaccines (YF-VAX�) [31], and wereshown to induce immune responses that replicated known humanimmune response profiles. Notably, using elderly donors, influenzavaccine-induced antibody responses were measured in theMIMIC�

system that replicated the well-recognized immunosuppressedstate of this donor population. Therefore, it is suggested that theMIMIC� system may serve as a sensitive and predictive in vitro testto simulate a clinical trial for a diverse population (using cells fromdonors selected for ethnicity, age, gender, etc.), prior to or inparallel with the initiation of animal studies, in which responsesmay differ significantly.

3.2. Assessing innate immunity induced by adjuvants using animalmodels

Ennio De Gregorio (Research Center, Novartis Vaccines andDiagnostics, Siena, Italy) reviewed the early immunological eventstriggered by the Novartis’MF59� vaccine adjuvant. MF59� is an oil-in-water emulsion containing squalene. It is a component oflicensed seasonal and pandemic influenza vaccines and was shownto elicit clinical benefit over non-adjuvanted influenza vaccines inthe overall population, including elderly subjects [32] and children[33]. The very large database existing today shows that MF59� isable to induce increased immunogenicity and breadth of the anti-body response [34], and exhibits low reactogenicity and excellentsafety profile at all ages, as shown by several controlled clinicaltrials [35] and by very large post-marketing surveillance [36].

In the past few years, the mode of action of MF59� has beenextensively dissected in mouse models. Analysis of the effect ofdifferent adjuvants localized in mouse muscle (by microarray andflow cytometric analysis) after intramuscular immunization iden-tified MF59� as a strong immune potentiator at the injection site,inducing genes involved in leukocyte transendothelial migration[37]. Flow cytometric analysis showed that MF59� triggers rapidblood cell recruitment into the injectedmuscle, including abundantneutrophils followed by inflammatory monocytes, eosinophils,dendritic cells and macrophages [38]. However, neutrophils did notappear to play a fundamental role, since their depletion did notalter MF59� adjuvanticity [38]. Using fluorescently-labeled MF59�

complete emulsion and ovalbumin (OVA) antigen, the majority ofcells recruited at the injection site were shown to take up boththe antigen and the adjuvant and efficiently transport the antigento the draining lymph nodes [38]. Furthermore, a microarrayanalysis performed both on muscle and lymph nodes of miceimmunized with different Toll-like receptor (TLR)-dependent orTLR-independent adjuvants showed that MF59� only up-regulatesgene transcription at the injection site [39] and not in draininglymph nodes. Therefore, it is suggested that MF59� has a localizedtissue fingerprint, eliciting dual adjuvant properties: combining

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antigen delivery with immune potentiating activity at the injectionsite and leading to the recruitment and activation of circulating andresident APCs. This discordance between immunomodulation ofresponses localized to the site of immunization and in the draininglymph nodes (where responding lymphocytes may be patternedwith homing receptors) introduces new complexities and oppor-tunities for the targeting of immune responses to discreteanatomical sites.

This concept was further explored by Arnaud Didierlaurent (GSKvaccines, Rixensart, Belgium) who reviewed the key features of themode of action of two GSK Adjuvant Systems (AS), AS03 and AS04.

AS03, which contains a-tocopherol and squalene in the form ofoil-in-water emulsion, has been considered for development ofpandemic and seasonal influenza vaccines, because, in non-clinicaland clinical models, it induces superior adaptive responses againstthe vaccine antigen, both in terms of antibody levels and immunememory [40]. In a comparative study with aluminum hydroxideadjuvant, AS03 was shown to promote a superior antigen-specificadaptive immune response, a feature associated with induction oftransient and local innate immune responses [41]. Following i.m.immunization, a specific signature of chemokines and cytokineswas detected at the injection site but in this casewas also paralleledby enhanced recruitment of monocytes and granulocytes indraining lymph nodes (dLNs) [41]. Moreover, using fluorescently-labeled antigen, monocytes were shown to constitute themajority of antigen-loaded cells in dLNs [41]. Although AS03 didnot directly activate dendritic cells [42] in vitro, AS03 injection alsoactivated and recruited DC in vivo, thus suggesting an indirectactivation of DC by the pro-inflammatory environment. It was alsonoted that the presence of a-tocopherol in the formulation isrequired for AS03-enhanced immunomodulatory properties andthat a spatio-temporal co-localization of AS03 with the antigen ismandatory, although not directly dependent on physical associa-tion with the antigen [41].

The Adjuvant System AS04 contains a nontoxic bacterial LPS-derivative, MPL (TLR4 agonist), and alum, and is used ina licensed hepatitis B vaccine [43] and human papillomavirusvaccine [44]. AS04 induces a transient and local cytokine responses,which stimulate the migration and activation of DC and monocytesin dLNs [45]. Conversely to AS03, AS04 induces DC activationin vitro but, similarly to AS03, the antigen and the adjuvant need tobe co-localized at the injection site to have a beneficial adjuvanteffect on relevant APCs, resulting in efficient stimulation of adap-tive T and B cell responses, including a high level of memory B cells[45]. Therefore, a common outcome of early immune activation byAS is the recruitment of efficient APCs in the dLN, which couldfurther support T cell and antibody responses.

A different aspect of lymph node targeting was explored byClaire-Anne Siegrist (WHO Collaborating Center for Vaccinologyand Neonatal Immunology, Geneva, Switzerland) who drewattention to the influence of antigen-adjuvant formulations, andthe relative timing of antigen or adjuvant exposure on the kineticsand breadth of APC targeting and activation in the dLNs. She sug-gested that vaccine efficacy largely depends upon APC activation,which then conditions specific CD4þ T helper cells to differentiateinto one of several lineages of effector cells. For instance, usingpromising candidate vaccines against tuberculosis, the antigen H1(Ag85B-ESAT-6) formulated in IC31� (Intercell) [46] or the cationicliposomal adjuvant CAF01 (Statens Serum Institute) [47], it wasdemonstrated that the effectiveness of the vaccine relied on pro-longed uptake by a minute lymph node Agþ adjuvantþ (Adj) DCpopulation. Yet, while injection at the same site but with separateadministration (in separate syringes) of the H1 antigen and theCAF01 adjuvant did not prevent combined Agþ Adjþ DC targetingand activation, it resulted in an overall reduced protective TH1/

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TH17 immune response [48]. Therefore, targeting the antigens andimmunomodulators to the same activated APCs is not sufficient toelicit protective TH1/TH17 vaccine responses.

To further identify the determinants of TH1/TH17 adjuvanticity,the in vivo delivery kinetics of several fluorescently-labeled anti-gens and adjuvant systems were modified and the targeted APCsidentified by flow cytometry [48]. The study revealed that it is theantigen that plays a crucial role in immune interference with TH1responses and thus defines the specificity of the immune response[48]. In fact, for TH1/TH17 responses to be elicited, early antigentargeting of DCs prior to their activation by adjuvant moleculesshould be avoided, otherwise non-activated antigen pulsed DCsrecruit antigen-specific naïve T cells and trigger their proliferationtowards TH2 rather than TH1/TH17 effector/memory cells [48].Therefore, it is suggested that vaccine formulations with minimal“leaking” of antigen, hence inducing synchronized DC activationand antigen exposure, should be carefully selected for the inductionof TH1/TH17 responses.

In summary, these presentations highlighted the varying effectsof different adjuvant types on injection site or draining lymph noderesponses and the way in which interactions in the timing andlocation of immune activation may result in differential immuneoutcomes. This offers both opportunities to refine immuneresponses, but also challenges in predicting eventual clinicaloutcomes from these complex interactions.

3.3. Biomarkers of innate immunity following vaccination: towardssystem biology

The use of systems biology approaches to guide rational vaccinedesign and development was covered by Bali Pulendran (EmoryUniversity, Atlanta, GA, USA). Systems biology is an interdisci-plinary approach that integrates, through computational analyses,multiple datasets from different biomic platforms, along withimmunological and clinical parameters [49]. By using thisapproach, early gene signatures for predicting immune responsesin humans vaccinated with the yellow fever vaccine YF-17D [50] orwith influenza vaccines [51] were identified. For instance, with theyellow fever vaccine, a novel early gene signature, includingcomplement protein C1qB and a critical player in the integratedstress response (EIF2AK4, also known as GCN2), was identified andpredicted CD8þ Tcell responses with a high degree of accuracy [50].In addition, it also identified a separate signature, the B cell growthfactor TNFRSF17 (BCMA), as a key predictor of neutralizing antibodyresponse [50].

Subsequent to these observations, several in vitro and in vivoexperimental approaches tested and verified the role of these toprovide further mechanistic insights. For instance, consistent withthe EIF2AK4 role for regulating protein synthesis in response toenvironmental stresses by phosphorylating the a-subunit of initi-ation factor 2 (eIF2a), it was demonstrated that YF-17D inducesamino acid deprivation-induced stress responses in dendritic cells,resulting in phosphorylation of eIF2a and formation of stressgranules [50].

Another example of predictive immune profiling has beendemonstrated for influenza vaccines in a study of healthy adultsvaccinated with a trivalent inactivated vaccine (TIV) [51]. Notably,expression levels of CaMKIV (a calmodulin-dependent proteinkinase involved in neural functions, stem cell maintenance, and Tcell development) at day 3 following vaccination with TIV wereshown to inversely correlate with antibody titers at the peak of theimmune response, an observation further validated using CaMKIV-deficient mice [51]. Therefore, the development of predictivesignatures for vaccine immunogenicity and efficacy using systemsbiology may provide significant insights into the mode of action of

Please cite this article in press as: Mastelic B, et al., Potential use of inflvaccine safety, Biologicals (2012), http://dx.doi.org/10.1016/j.biologicals.2

vaccine-induced immune responses, and may serve as usefulbiomarkers for predicting the efficacy of novel vaccine candidates,or for predicting and understanding some vaccine adverse reac-tions or suboptimal (reduction in efficacy) immune responsesamongst a high risk population, such as infants or the elderly.Deeper insights into the mechanisms underlying efficacy or reac-togenicity may also guide rational and directed approaches toenhancing the former while mitigating the latter, in a break fromthe usual empirical approach to vaccine design.

3.4. Practical application of biomic platforms for retrospectiveanalysis of adverse reactions

Wei Zhu (MedImmune, Gaithersburg, MD, USA) discussed theapplication of human whole-genome microarrays to capture tran-scriptional variations among subjects following vaccination. Acomparative study on peripheral whole blood from children (12e35 months) vaccinated with Live Attenuated Influenza Vaccine(LAIV) or trivalent inactivated vaccine (TIV) was conducted andidentified many differentially expressed genes, with LAIVrecipients showing particularly greater variation in the transcriptprofile [23]. For practically useful techniques to emerge from thecomplex datasets generated in research studies, a degree of stan-dardization and “coarse-graining” of results will be required. Onesuch method was already described by I-Ming Wang (Section 2.3)using functionally-related gene-modules. Wei Zhu reported anapproach in which genes with similar transcriptional profiles weregrouped using a hierarchical clustering, which revealed 6 co-expression gene clusters. As many of the co-expressed genesoften have related functions and are involved in similar pathwaysor gene networks, it revealed many genes commonly induced byviral infection and playing essential roles in type I IFN signaling,antigen presentation, cellular and humoral immune responses [23].The LAIV was shown to induce higher expression of type I IFNsignature genes than TIV 7 days post-vaccination, which mayexplain some evidence of LAIV-induced protection againstinfluenza-like illness in the first two weeks post-vaccination [23].However, it should be noted that the samples were collected ata single and late time point (day 7) following vaccination and thusmay not best capture innate immune responses. Therefore, it issuggested that blood samples should be collected at earlier timepoints and prior to each vaccination, to better reflect any changes inimmune responses. Indeed, early standardization in the protocolsfor evaluating kinetics of gene expression, as well as standardizedgene clustering in the analysis, was noted to be crucial to ensurecomparable data is available from the various studies and tomaximize the prospects of identifying common, importantbiomarkers.

Notably, the activation of type I IFN pathway by LAIV was alsodemonstrated in a recent study from Nakaya et al. using thesystems biology approach [51]. Therefore, measuring changes intype I IFN or type I IFN-regulated genes after vaccination could bean important biomarker identifying early responses to LAIV.Whole-genome microarrays allow the measurement of broad-spectrum pathway changes following vaccination, and it was sug-gested they be used in parallel with other techniques to obtaina fuller profile of immune responses and to identify more specificpredictors of vaccine efficacy.

Eugene Maraskovsky (CSL, Parkville, Australia) discussedanother example of retrospective analysis of adverse reactions,particularly the investigation into the cause of TIV-induced AE inthe 2010 pediatric population. In fact, during the 2010 SouthernHemisphere influenza season, there was an unexpected increase inthe number of febrile seizures reported predominantly in childrenunder the age of 5 within a day of vaccinationwith the CSL 2010 TIV

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vaccine [52]. However, it should be noted that there was a com-plete change in the composition of strains between 2009 (A/Bris-bane/59/2007, A/Uruguay/16/2007, B/Florida/4/2006) and 2010 (A/California/7/2009TIV, A/Wisconsin/15/2009, B/Brisbane/60/2008)TIVs. Therefore, a comparative study with various TIVs (previousseasons’ or comparator 2010 licensed vaccines, as well as re-engineered TIVs) was conducted to identify vaccine componentsor surrogate markers that may have implications for these AEs,using several methods; whole blood (adult and pediatric) in vitrocytokine profiling, mammalian cell lines, microarray-based geneprofiling and in vivo studies using rabbits, ferrets, new born rats andnon-human primate animal models. The in vitro stimulation assaysand gene expression profiling of adult and pediatric whole blood aswell as the TIV-immunized NHP blood, revealed that CSL TIVs ingeneral, and in particular the CSL 2010 TIV, induced higher levels ofcytokines, greater changes in gene signatures and higher serumhemagglutinin inhibition activity than comparator 2010 TIVvaccines. Interestingly, replacement of the H1N1 pandemic A/Cal-ifornia/07/2009 strain in the 2010 TIV with another H1N1 strain,altered the cytokines/chemokines and gene signatures, in partic-ular by increasing gene signatures involved in immune regulation.This suggested that the A/California/07/2009 strain may not havebeen engaging “breaks on the system genes” as effectively asprevious H1N1 strains. The induction of these signals was associ-ated with a heat-labile, viral-derived component. Using theHEK293 cell line expressing the NF-kB reporter, it was demon-strated that heat pre-treatment (>65 �C) and increases in thepercentage of the sodium taurodeoxycholate (TDOC) detergent,used to disrupt influenza virus to make a split virion vaccine, allquenched NF-kB signals. Interestingly, all recent CSL TIVs contain-ing B/Brisbane/60/2008 (2009/10 NH season) induced NF-kBsignaling in the HEK 293 NF-kB reporter cell line and the firstintroduction of this B strain in the 2009/10 NH seasonwas found tobe associated with increased mild/moderate clinical fever in theyoungest children. Therefore, it was suggested that B/Brisbane/60/2008 likely sensitizes for increased mild/moderate fevers (corre-lating with the clinical observations), and that the combination ofA/California/7/2009 with B/Brisbane/60/2008 into CSL 2010 TIVfurther increased these fevers triggering febrile seizures ina portion of the youngest children. Thus, the observed AE mayresult from complex and multi-factorial causes, which likelyinvolve the new viral strains used for the 2010 TIV and how CSL’smanufacturing process preserved the pyrogenic characteristics ofthese viral-derived components. Hence, the use of these differentbiological assays may assist in the further evaluation of CSL’smanufacturing process and may be useful in minimizing the futureincidence of such febrile reactions in the pediatric population.However, the implementation of these complex assays withinthe tight manufacturing time-tables of seasonal TIV will bechallenging with in vitro surrogate assays likely to be the mostfeasible. Consideration may also need to be given to pre-screeningprototypic influenza vaccine strains to assemble the least reacto-genic isolates in advance using these biological assays.

4. Monitoring vaccine effects on unrelated immuneresponses

Although rare, serious adverse events may occur after vacci-nation and are generally unlikely to be detected in preclinicalevaluation because of their low frequency and the limitednumbers of animals that can be used in testing. Currently, pro-longed post-marketing monitoring is recommended to identifyand report any infrequent and serious adverse events aftervaccination. However, the tools are now available to further studyand perhaps identify early biomarkers that would correlate and

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predict rare events such as atopy, autoimmunity and immuno-logical interference.

4.1. Innate immunity biomarkers to predict autoimmune diseases

As stressed by Willem van Eden (Utrecht University, TheNetherlands) there is a challenging need for development of care-fully designed in vivo models for vaccine safety analysis. Indeed,innate immunity is known to have an important role in generatingrobust and long-lasting adaptive immune responses, crucial tovaccine effectiveness. However, innate immunity also plays a crit-ical role in the induction or regulation of autoimmune diseases [53],as recently emphasized by the recognition of gut microbiota(commensal bacteria) as a factor in the development of autoim-munity in animal models [42]. For instance, a protective effect ofmicrobiota colonization was demonstrated in influenza [54] and inthe development of autoimmune diabetes in non-obese diabeticmice [55], whereas, in experimental autoimmune encephalomy-elitis (EAE) the commensal gut flora was causally involved with thedisease [56]. Other innate immune factors that may play a criticalrole in triggering autoimmunity include microbial heat shockproteins (Hsp) [57], proteasomes [58] and TLR ligands [59,60].For instance, heat shock proteins are produced under stressfulconditions, including tissue damage or inflammation, and weredescribed as having immunoregulatory potential to maintainhomeostasis following tissue damage in several disease models[61]. However, it was recently shown that Hsp also tolerized DC andinduced anti-inflammatory regulatory T cells [57]. Vaccinationinduced inflammatory response may also lead to the induction ofimmunoproteasome formation, which could result in altered Agprocessing within proteasome, thus altering epitope selection andlead to autoimmunity [58]. Finally, TLR triggering by CpG was alsoshown to convert T cell autoreactivity into overt autoimmunediseases, inducing disease relapses in EAE [59]. Thus, risk of seriousadverse events may not be detectable in in vitro systems or withnew technologies without well characterized and well chosenin vivomodels to study the complex immune behavior in responsesto vaccines. The commensal microbial pressure adds considerablevariation to animal models and additional complexity in setting upan experimental model best representative of humans, includingmajor differences between mice and human immunology [62], inorder to assess any risks of autoimmunity. A potential experimentwas suggested to probe silent autoimmunity by vaccine- oradjuvant-induced pro-inflammatory responses, using transfermodels of titrated autoantigen TcR transgenic T cells (activation of“silent auto-reactivity ”) [60].

4.2. Early autoantibodies as biomarkers to predict autoimmunediseases

George Eisenbarth (Barbara Davis Center for ChildhoodDiabetes, University of Colorado, USA) discussed the value ofearly autoantibodies as predictive biomarkers of autoimmunediseases. The clinical onset of autoimmune diseases is oftenpreceded by an asymptomatic state with detectable autoanti-bodies. For instance, type I diabetes is a striking example ofa disease in which autoantibodies serve as a predictive marker.Type I diabetes is an organ-specific autoimmune disease, whichresults in pancreatic islet cell destruction, reflected by autoan-tibodies to islet cells, insulin, IA-2 (Insulinoma AssociatedAntigen), ZnT8 (Zinc Transporter 8), and to glutamic decar-boxylase. Islet autoantibodies appear early in life, and testingfor multiple antibodies identifies unaffected individuals at veryhigh risk of type 1 diabetes. Therefore, specific assays forautoantibody detection, including solid-phase and fluid-phase

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radioassays, were developed and achieved high sensitivity,allowing prediction of type 1 diabetes and detection of thelatent autoimmune diabetes in adults [63,64]. However,some children developed persistent insulin autoantibodieswithout evidence of progression to diabetes, even though theyappeared positive for autoimmunity using the standard assay.Therefore, a new electrochemiluminescent nonradioactivefluid phase autoantibody assay, that detects anti-insulinautoantibodies with better specificity and sensitivity thanstandard radioassays, has recently been developed, and hasdemonstrated higher specificity in predicting the risk of diseaseonset [65].

The development of autoreactive Tcell assays that would predicta high risk of autoimmune disease before vaccination represents animportant goal to achieve. Meanwhile, pre-vaccination sera shouldbe available for post-trial analysis of immune sera in case anyclinical signs appear, thus distinguishing vaccine-induced from pre-existing autoreactive antibodies. However, it should be noted that itwill be difficult to exclude vaccine-induced acceleration orprogression of autoimmunity for individuals with pre-vaccineautoantibody titers.

4.3. Biomarkers to predict effects of vaccination in atopic children

Patrick Holt (Telethon Institute for Child Health Research,West Perth, Australia) presented a genomic-based approach thatmay help predict vaccine safety and immunogenicity in children,particularly in atopic children. By using a systems biologyapproach along with a weighted gene co-expression networkanalysis, differential induction of gene networks was observedcomparing TH-memory responses induced by a TH2-polarizedvaccine and allergen-specific TH2-associated responses associ-ated with allergic disease [66]. Briefly, PBMC from preschoolchildren vaccinated during infancy with the diphtheria-tetanus-acellular pertussis (DTaP) vaccine, or from atopic childrenresponsive to house dust mite (HDM) allergens, were culturedfor 24h with the appropriate antigens. Subsequently, genenetworks expressed during reactivation of TH-memory cellsstimulated with DTaP antigen were compared with memoryresponses to HDM allergens in sensitized children. This strategyallowed the identification of a disrupted equilibrium betweenTH1 and TH2-associated gene networks in atopic children [66].In fact, the pertussis-specific memory response to DTaP vaccineswas shown to include a TH2 module, which was counter-balanced by parallel networks encoding pathways associatedwith TH1 and type I IFN (antimicrobial) mediated immunity.However, although the TH2-polarized HDM response closelyresembled the pertussis-specific TH2 network module, in anoverall atopic response network the TH1 module was notpresent and the antimicrobial module was largely down-regulated with allergic inflammation.

It is known that DTaP vaccine-responders can in somecircumstances exhibit an atopy-like TH2 phenotype associatedwith strong boosting of vaccine antigen-specific IgE titers, andwith local reactions at the booster injection site [67]. As a conse-quence, concerns have been raised regarding the potentialcontribution of the TH2 component of memory responses inchildren to side effects that may be induced by other (includingfuture) TH2-polarizing vaccines, particularly amongst atopic chil-dren who exhibit a generalized TH2 “bias” in their immuneresponses. The use of genome wide profiling in conjunction withgene co-expression network analysis provides a novel approach tothe early detection of inflammatory pathways triggered by vacci-nation which may predict potential induction of pathogenicreactions in the host.

Please cite this article in press as: Mastelic B, et al., Potential use of inflvaccine safety, Biologicals (2012), http://dx.doi.org/10.1016/j.biologicals.2

4.4. Biomarkers to predict bystander immunological activationfollowing vaccination

Non-antigen specific “bystander” activation of immune cellsmay occur during an immune response due to diffusion of che-mokines and cytokines from antigen-activated cells in the locality.A more florid immune activation may result in higher levels ofcytokines and chemokines and therefore increased bystanderactivation. Gianfranco Di Genova (Cancer Sciences Unit, Faculty ofMedicine, University of Southampton, UK) pointed out that earlymonitoring of bystander T cell activation may allow more accurateinterpretation of vaccine-induced immunity and thus better predictthe effectiveness of a vaccine. During an Ag-specific T cell response,it was shown that bystander activation of T cells, including CD8þ

and CD4þ T cells, may occur. It was exemplified by stimulation ofheterologous T cells by cytokines [68] or TCR-independentsignaling. Mouse models of viral, bacterial or parasitic infections,revealed proliferation of T helper memory (Thmem) cells [69],which may protect or cause pathology and thus subvert the hostresponse to subsequent vaccination. In humans, vaccine-inducedbystander events were demonstrated in healthy subjectsfollowing recall vaccination with tetanus toxoid (TT) [70]. Thmemcell response to TT was accompanied by parallel expansion ofThmem cells (but not of naïve T cells) and cytokine productionspecific to two common unrelated antigens: purified proteinderivative of tuberculin (PPD) and Candida albicans [70]. While theantibody production against TT was boosted approximately 2weeks after vaccination and then gradually declined, pre-existingantibodies against the two unrelated antigens remainedunchanged, hence emphasizing the need for the antigen forsubsequent specific antibody responses. These observations werevalidated and further investigated in a preclinical mouse model[71]. This model used activated OTII CD4þ T cells specific for oval-bumin, which were transferred into mice pre-vaccinated with TT. Aboost of the TT-specific memory response in the recipients led tobystander proliferation of the transferred unrelated OTII T cellsand to the identification, in vitro, of IL-2 and IL-7 as potentialmediators [71].

In healthy human subjects who received a full course of primaryhepatitis B vaccination, preliminary data indicated that bystanderactivation of CD4þ T cells against three unrelated antigens (TT, PPDand Candida albicans) occurs after priming, and appears to correlatewith the strength of the hepatitis B-specific antibody response.Therefore, early assessment of bystander T cell activation alongsidevaccine-specific immunity could be very informative in predictingbroader vaccination outcome.

5. Which biomarkers should be considered for early vaccinetrials?

This question was considered by a panel made up of represen-tatives from industry, academia and regulatory authorities. It wassuggested that biomarkers should predict relatively commonevents such as inflammatory reactogenicity that may not beevident in preclinical models, and ideally, rare vaccine-relatedsevere adverse events, such as autoimmune diseases, for whichno animal model may exist and very large human populations mayneed to be studied over long periods. Rare adverse events areacquiring greater importance in the public opinion and mayundermine vaccine acceptability. However, the panel promptlyacknowledged that, in the context of vaccine development, it isunlikely that a single “universal” marker will be found that coversevery demand and is predictive for every vaccine candidate basedon formulation, pathogen and host variability. Therefore, the searchshould rather concentrate on a combined biomarker signature for

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each vaccine and specific population. Nevertheless, with currenttechnology and resources, we cannot yet achieve personalizedpredictive markers. We should instead, by searching for thresholdbiomarkers common to different signals, focus on the developmentof new vaccine and adjuvant candidates with optimal benefit-riskratio for use at a broad population level.

With integrated biomic platforms (genomics, transcriptomics,proteomics and metabolomics) there are prospects for unravelingand selecting new biomarkers that could contribute to subject-specific AEs, or affect the quality of immune response in bothanimal models and humans and thus may improve safety evalua-tions in preclinical studies and vaccine trials in humans. However,in the field of transcriptomics, at present, the comparability ofstudies is limited due to the variety of methods employed in thedetection and computation of gene expression differences andgenetic signatures. Meanwhile, valuable additional data may beobtained from animal models and human cell lines. It was sug-gested that concerted efforts were needed to move the fieldforward from conventional approaches for early safety and efficacytesting of a vaccine, by standardizing and integrating new tech-nologies now available for non-clinical studies. In fact, the practicalvalue of several alternative reproducible and predictive in vitromethods, including MAT, MM6 and MIMIC� have now beenconfirmed and these methods are available for gaining greaterinsight into pyrogenicity and cell-mediated immunity. For instance,as pointed out by William Warren, the in vitro system MIMIC�

enables evaluation of human immune responses against candidateadjuvants and vaccines, and could thus be considered for immu-notoxicity assessment, such as endothelial cell damage anddisturbance of vascular integrity, or the induction of an exaggeratedinnate immune response that may be predictive of a cytokine“storm”. If the predictive value of such assays can be confirmed,they may enable meaningful comparisons between differentdatasets and complement in vivo animal studies to select potentialvaccine candidates for preclinical evaluation. However, it isimportant to establish cut-off or threshold values that can distin-guish between safe vs. unsafe products and to include internalcontrols and standards of vaccines and adjuvants with knownreactogenicity profiles to demonstrate assay consistency andpredictive value.

Nathalie Garçon (GlaxoSmithKline (GSK) Biologicals, Rixensart,Belgium) further discussed in vitro and in vivo comparativeassessment of adjuvants and formulations. Despite significantdifferences between mice and humans [62,72], in both the innateand adaptive arms, the use of animal models in preclinical studiescannot yet be bypassed. Indeed, animal models are particularlyuseful for unraveling the vaccine adjuvant action mechanisms,measuring changes in immunological parameters, and for evalu-ating toxicology, thus providing guidance for Phase I clinicalstudies. However, a predictive preclinical model for one givenadjuvant is greatly dependent on vaccine formulation and may notbe applicable across all species (rodent/non-human primates andhumans), hence being misleading across adjuvants. For instancethe recent study of Morel et al. [41], investigating the key featuresof AS03mode of action, well illustrates combined in vivo approachesin mice and ex vivo approaches in human cells, as well as theimportance of testing adjuvant formulations in vivo to determinehow formulation influences adjuvant activity. In vitro immu-noactivities of adjuvants do not always predict or correspond to theimmunomodulatory activity of adjuvant formulations in vivo.Therefore, in vitro studies should be complemented by in vivostudies, preferably involving the most relevant animal model,rather than amodel of convenience. Suchmodels may reflect in vivocell-fluid and cellecell interactions whereas the cellular environ-ment may be difficult to replicate in vitro.

Please cite this article in press as: Mastelic B, et al., Potential use of inflvaccine safety, Biologicals (2012), http://dx.doi.org/10.1016/j.biologicals.2

One recommendation from regulatory agencies would be toestablish a more comprehensive program for blood collection,storage and archiving of clinical data, and in particular, a longerfollow-up of subjects, whichwould be helpful if AE emerged at latertime points. By using the new biomic platforms, this sample“banking” would allow safety profile comparison across a pop-ulation or between specific groups (e.g., vaccinated subjects vs.patients recovering from infection) and enable the future discoveryand validation of clinically relevant biomarkers for preventivevaccines. Various contributors commented on the hurdles placedby ethical considerations and legislation restricting the use ofhuman tissue in achieving this aim, especially from clinical trialswhich may have very defined endpoints. However, such samplebanking may be feasible under well designed pre-licensure or post-marketing studies approved by IRB with signed patient consentforms.

As the relationship between vaccination and adverse events isoften “casual rather than causal”, it is suggested that an improvedpost-marketing surveillance program should be established,resulting in an enlarged database, which would help in identifyinghigh-risk subjects and perhaps finding any biomarkers associatedwith potential adverse event outbreaks. Meanwhile, it was sug-gested that screening for potential “atopy-like” properties bygenome-wide expression profiling/network analysis should beconsidered, or, at least, it may be worth considering measuring IgEresponses in vaccinated subjects, as it could assist in demonstratingthe presence or absence of atopy. In fact, this would allow cir-cumventing the use of “TH2-bias” adjuvants in these high-riskpopulations. William Warren also pointed out that there are newbioplex bead array systems that allow antibody forensics andshould be considered for regular assessment of autoantibodies,immune interference and cross-reactivity. By using these differentavailable tools, a solid database could be built and allow earlyidentification of high-risk subjects and biomarkers associated withrare adverse events, the experience of which may discouragepeople to get vaccinated.

6. Conclusion

Although the identification of potential biomarkers forpreclinical safety evaluation of a vaccine is currently a work inprogress, the use of such identified biomarkers still needs to proveits robustness in clinical trials. However, it is hoped that oncedemonstrated to be predictive, biomarker assays could provide newtools for predicting reactogenicity of vaccine candidates. Several ofthese new biomarkers have been successfully applied retrospec-tively to evaluate and explain observed reactogenicity of marketedvaccines, such as febrile episodes and convulsions associated withsome influenza vaccines. It was acknowledged that the FDA,academic researchers and pharmaceutical companies shouldcollaborate to standardize and demonstrate the validity of thesenew approaches in order to have cross-study comparable data.Such an effort will enhance the introduction of these validatedbiomarker-based methods to complement current assessmentstrategies for safety and effectiveness of a specific vaccine.However, it will still require a mighty effort to “personalize” theseassays for specific targeted subpopulations at specific doses; hencethe need to first perform these assays on vaccines with establishedsafety profiles. Finally, the integration of these new biomarkerswith traditional correlates of safety (such as biochemistry andhematology panels and clinical symptoms and signs) will be chal-lenging, as the new techniques tend to expand and refine under-standing of the mechanisms underlying reactogenicity and efficacyrather than replace existing assays. Learning how to best use, andnot abuse, the flood of data from these powerful new tools will

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require years of careful preclinical and clinical research, standard-ization, and co-operation.

Acknowledgments

We thank Betty Dodet (Dodet Bioscience) for her skillful help inthe preparation of this report. This workshop was made possible byunrestricted educational grants from GlaxoSmithKline Biologicals,Sanofi Pasteur, Novartis, IMI, Pfizer and CSL.

Appendix A. List of participants

Paul-Henri Lambert, University of Geneva; William Egan,Novartis; Ian Gust, University of Melbourne, Australia; RebeccaSheets, NIAID/NIH, USA; Johannes Löwer, IABS, Germany; EickeLatz, University of Bonn & University of Massachusetts, Germany &USA; Jeffrey Roberts, FDA/CBER/OVRR, USA; Ingo Spreitzer, Paul-Ehrilich-Institut, Germany; Ennio De Gregorio, Novartis, Italy; I-Ming Wang, Merck Research Laboratories, USA; Alessandra Mor-tellaro, Singapore Immunology Network (SIgN); Agency for Science,Technology and Research (A*STAR), Singapore; Norman Baylor,Biologics Consulting group, USA; Bali Pulendran, Emory University,USA; William Warren, VaxDesign Campus, Sanofi Pasteur, USA;Claire-Anne Siegrist, University of Geneva, Switzerland; ArnaudDidierlaurent, GSK Biologicals, Belgium; Eugene Maraskovsky, CSL,Australia; Wei Zhu, MedImmune, USA; Robert Coffman, DynavaxTechnologies, USA; Pieter Neels, European Medicines Agency, UK;Hana Golding, FDA, USA; Nathalie Garçon, GSK Biologicals,Belgium; Willem van Eden, Utrecht University, The Netherlands;Patrick Holt, Telethon Institute for Child Health Research, Australia;Gianfranco Di Genova, University of Southampton, UK; GeorgeEisenbarth, Barbara Davis Center, University of Colorado, USA;Michel Goldman, Innovative Medicines Initiative, Belgium; DavidLewis, University of Surrey, UK.

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