Predictive markers of safety and immunogenicity of adjuvanted vaccines

Biologicals xxx (2013) 1e11

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Meeting report

Predictive markers of safety and immunogenicity of adjuvanted vaccines Beatris Mastelic a, *, Nathalie Garçon b, Giuseppe Del Giudice c, Hana Golding d, Marion Gruber e, Pieter Neels f, Bernard Fritzell g a

WHO Center for Vaccinology and Neonatal Immunology, University of Geneva, CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland GlaxoSmithKline Vaccines, Rixensart, Belgium Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy d Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892-0001, USA e Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Rockville, MD, USA f Federal Agency for Medicines and Health Products, EMA e CHMP, Belgium g IABS Human Vaccines Committee, France b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 August 2013 Received in revised form 29 August 2013 Accepted 31 August 2013

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.

Keywords: Vaccine Adjuvant Vaccine safety

1. Introduction Abbreviations: AEs, adverse events; APCs, antigen presenting cells; AS, adjuvant systems; BNP, induced bovine neonatal pancytopenia; BM, bone marrow; BVD, bovine virus diarrhea; CRP, C-reactive protein; DDCs, dermal dendritic cells; Env, envelope glycoprotein; FDA, Food and Drug Administration; GFPDL, genome-fragment phage display libraries; HA, hemagglutinin; HAI, hemagglutination-inhibition; HBsAg, Hepatitis B surface antigen; HBV, Hepatitis B virus; HIV-1, human immunodeficiency virus type 1; ID, intradermal; IM, intramuscular; IFN, interferon; ISCOM, immune stimulating complexes; LAIV, Live Attenuated Influenza Vaccine; LC, Langerhans cells; LPS, lipopolysaccharide; MIV, monovalent inactivated vaccine; Mo, human primary monocytes; MoA, mode of action; MM6, Mono Mac 6; MPL, 3O-desacyl-40 -monophosphoryl lipid A; MVA, modified vaccinia ankara; mPGES1, microsomal PGE synthase-1; nAbs, neutralizing antibody; NA, neuraminidase; NHP, non-human primates; PBMC, peripheral blood mononuclear cells; PGE2, prostaglandin E2; PLA, poly-lactic; rAd5, replication-defective adenovirus 5; SC, subcutaneous; SPADE, spanning-tree progression analysis of density normalized events; SPR, surface plasmon resonance; TC, transcutaneous; TFH, T follicular helper cells; TIV, trivalent inactivated vaccine; TLR, toll-like receptors; WHO, World Health Organization. * Corresponding author. Tel.: þ41 22 3795781; fax: þ41 223795975. E-mail address: [email protected] (B. Mastelic).

A workshop called “Predictive markers of safety and immunogenicity of adjuvanted vaccines”, organized by the International Association for Biologicals (IABS), was held at the U.S. Pharmacopeial Convention (USP) from 18 to 19 April 2013 in Rockville, Maryland. The goal of this conference was to bring together a group of scientists from academia, regulatory agencies and industry with wide expertise to discuss where the adjuvant field stands, and provide guideposts for future development of effective adjuvants and adjuvanted vaccines, and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. Hana Golding (Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Division of Viral Products, Bethesda, USA) introduced the meeting by reviewing what had been learnt from recent experience in the adjuvant field and outlined the challenges ahead. Aluminum salts have been used for decades as the only acceptable adjuvants for vaccines, although the

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precise mode of action (MoA) by which they augment the immune response continues to elude scientists. The need to appropriately increase the immunogenicity of many new antigens of interest that are poorly immunogenic combined with advances in cell biology and immunology has encouraged the search for new adjuvants. New adjuvants were also considered to enhance protection in immunologically hyporesponsive populations (immunocompromised individuals, newborns or the elderly) and dose sparing. Although a number of novel adjuvants have been under development for several decades [1], very few have been licensed owing to safety concerns. In most cases, increased adjuvant potency is associated with increased reactogenicity and toxicity. Indeed, because of intrinsic tissue irritant effect or ability to potently activate a large number of innate immune cells, resulting in high levels of pro-inflammatory cytokines and vasoactive substances, adjuvants may contribute to enhanced local reactogenicity and systemic toxicity. Despite usual resolution over time, these side effects remain an important barrier for licensure of adjuvanted vaccines and better acceptance of routine prophylactic vaccination in the community. Thus, as stated by Rappuoli et al. [2], “the development of vaccine adjuvants for human use has been one of the slowest processes in the history of medicine”. For instance, aluminumbased adjuvants were the only adjuvants used in vaccines approved for use in humans by the FDA until 2009, when the AS04adjuvanted vaccine against human papillomavirus vaccine was approved. In other countries, including European countries, novel adjuvants (MF59, a component of seasonal and pandemic influenza vaccines FluadÒ and FocetriaÒ; AF03, a component of pandemic influenza vaccine HUMENZAÒ; AS03, a component of pandemic influenza vaccine PandemrixÒ and ArepanrixÒ; and AS04, a component of hepatitis B vaccine FendrixÒ and human papillomavirus vaccine CervarixÒ) have been introduced in the formulation of new licensed vaccines [3]. Therefore, a major challenge in adjuvant development is to achieve a potent adjuvant effect while controlling reactogenicity or toxicity. On that premise, the workshop discussed the current state of the art in the adjuvant field, the lessons that can be learned from various clinical and non-clinical studies, and the significant hurdles to be overcome in designing adequate safety studies: the complexity of the immune system and the MoA of many new adjuvants, the lack of suitable experimental models and standardized predictive methods, the difficulties in assessing certain adverse events (AEs), and pre-existing genetic variations in the innate system of individuals. The promise and challenges of applying new technologies including system biology and genomics in the search for early biomarkers of adjuvant activity and safety signals were one of the main topics of the workshop. 2. Lessons from adjuvant comparative studies in humans and in non-human primates Several comparative studies with adjuvanted and unadjuvanted preparations have been conducted to demonstrate the clinical benefit of incorporating any adjuvant into vaccines. Joe Francica (VRC/NIH, Bethesda, Maryland, USA) presented a study conducted in nonhuman primates (NHP) on the effectiveness of different adjuvants and formulations in eliciting increased breadth and quality of immune responses to a human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogen, the HIV-1 clade-C trimeric gp140 envelope protein. In fact, one of the hurdles in developing vaccines against highly variable viruses, such as HIV1, is the relatively poor immunogenicity of the subunit Env glycoprotein antigens, which fail to elicit antibody responses that protect against the majority of circulating viral strains. Therefore, the use of adjuvants that incorporate agonists of toll-like receptors (TLR) may improve Env-specific humoral immunity. Indeed, the use of TLR

agonists as promising vaccine adjuvants is actively pursued, and some have reached advanced human trials and even registration [4]. Hence, the trimeric HIV-1 gp140 antigen was administered to NHP in a prime-boost regimen, either alone or formulated in a single adjuvant (MF59 or alum), or in combination with different TLR ligands [alum  a TLR4 or TLR7 agonist, MF59  a TLR4 or TLR7 agonist, or poly I:C (TLR3-ligand)]. Gene expression profiling of peripheral blood 24 h post-vaccination revealed that TLR agonists mediate distinct molecular signatures of early innate responses. Remarkably, the addition of TLR7 agonists to alum or poly I:C alone elicited higher and durable Env-specific binding and neutralizing antibody (nAbs) titers against a panel of tier 1 HIV viruses as compared to alum alone. However, the titers were equivalent to those obtained with MF59 alone. The addition of TLR agonists to MF59 did not improve the immunogenicity of the vaccine. Likewise, higher levels of HIV-specific antibody-dependent cellular cytotoxicity titers were induced with alum þ TLR7 agonist, MF59 or Poly I:C. Env-specific binding antibodies were also elicited at higher levels at the rectal mucosa by co-adjuvantation with TLR agonists. Notably, it was shown that immunization with alum þ TLR7 agonist elicited nAbs that target the CD4 binding site, a critical site of vulnerability on the viral surface. Consequently, a new assay was developed to gain further insight into the mechanisms of these improved Env-specific B cell responses, to establish qualitative differences and to identify and sort these HIV Envspecific B cells. Antigen-specific B cells (IgGþ IgM gp140þ cells) were sorted and the Ig loci characterized by 454 deep sequencing: a rapid, high-throughput sequencing technique with the aid of barcoded primers, which permits a substantial number of sequences from independent samples to be simultaneously analyzed [5]. This technique should be useful in future genetic assessment of B cell responses and may provide insight into the development of the high affinity antibody response. These studies were undertaken to investigate improvements to the magnitude and quality of immune responses to HIV Env that may be achieved with adjuvants such as MF59 or various TLR agonists. The benefits reported here may be necessary for the generation of an effective preventive HIV vaccine, but are likely not sufficient. Next-generation adjuvants should be leveraged along with other improvements to rational immunogen design or vaccine delivery to better guide immune responses towards the generation of broadly-neutralizing antibody responses. Arnaud Didierlaurent (GlaxoSmithKline Vaccines, Rixensart, Belgium) reported the results of a phase II randomized study (NCT00805389) designed to compare and characterize the innate and adaptive immune responses, as well as reactogenicity profiles induced by various GSK Adjuvant Systems (AS) (i.e. AS01, AS03 and AS04), in combination with the Hepatitis B surface antigen (HBsAg). As the use of adjuvant in vaccines is often associated with increased local or systemic reactogenicity [6], such comparative study of different classes of adjuvants might help unravel the complexities of immunological mechanisms behind this association. The study was conducted in healthy, Hepatitis B virus (HBV) naïve adults (18e 45Y, 140 individual per group). Subjects were allocated randomly (1:1:1:1:1) to one study group to receive by i.m. injection, at day 0 and 30, 20 mg of HBV surface antigen (HBs) adjuvanted with AS01B, AS01E (1/2 dose of AS01B), AS03A, AS04 (FENDrixÔ). Alum (Engerix-BÔ) was used as a benchmark adjuvant. Immunization with HBs adjuvanted with AS was safe and well tolerated. Overall, AS induction of antigen-specific CD4 T cell was associated with a higher prevalence of reactogenicity symptoms (AS01, AS03 > AS04 and alum). In the groups with the highest reactogenicity, transient increases in serum inflammatory cytokines (IL-6, IP-10 and IFN-g) and C-reactive protein (CRP) levels were accompanied by rapid and transient recruitment of neutrophils and monocytes, an observation consistent with preclinical models [7,8]. Some changes

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in innate parameters and incidence of reported systemic symptoms (such as fever) were more pronounced post-dose 2, suggesting that antigen-specific immune responses may have impacted innate immunity. A preliminary analysis performed on the HBsAg/AS01B group (with the highest fever incidence) to define potential associations between inflammatory biomarkers and fever was presented. No association was found between circulating proinflammatory cytokines and fever, as circulating cytokines seemed necessary but not sufficient to explain fever induction in all individuals. A multi-parametric modeling of the innate immune parameters was required to better discriminate vaccine-related fever from non-fever cases after the second vaccination. Finally, comparisons with similar studies conducted in NHP revealed that blood monitoring of immune responses may not be representative of the vaccine response occurring locally at site of injection and at draining lymph node. Remarkably, with ½ dose of AS01B, reduced reactogenicity was observed without affecting the magnitude of the T cell responses, suggesting that reducing adjuvant dose may decrease reactogenicity without affecting immunogenicity. Yet, it should be noted that the impact of reduced adjuvant dose observed in this study may only apply to this specific setting (naïve adults) and should not be generalized without further investigation. In fact, as pointed out by Heather Davis (Pfizer Vaccines Research, Ottawa, Canada), in different settings, AEs may be related to different vaccine constituents, such as the nature and the dose of antigen, and not to the adjuvant. For instance, a clinical trial conducted in healthy volunteers to evaluate the potential of CpG 7909, a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs, as an adjuvant to Engerix-BÔ (hepatitis B vaccine), revealed no significant difference in the incidence rate of local and systemic AEs with escalating doses of CpG (0e1 mg), all vaccines being well tolerated. In contrast, the addition of CpG 7909 to a malaria vaccine was associated with higher severity of local and systemic AEs [9]. Thus, for a specific adjuvant, discordant reactogenicity patterns may be observed when combined with different antigens. It does not appear acceptable to extrapolate reactogenicity of an adjuvant in a given antigen/adjuvant formulation to another one. 3. New paradigm in preclinical toxicity testing Potential adverse effects of drug candidates are evaluated following well-established guidelines on toxicology and safety pharmacology studies. Both include a core battery of tests conducted in vitro and in animals that allow the definition of the First In Human (FIH) dose for phase 1 clinical trials before drug testing in humans. However, for vaccines, several pitfalls were identified in the application of risk assessment guidelines. As pointed out by Marc Pallardy (Faculty of Pharmacy, Paris-South, France), current guidelines for vaccine pre-clinical safety evaluation are hindered by the extreme diversity of vaccine formulations, which includes the type and relative amounts of antigens and adjuvants incorporated in the vaccines, as well as the vaccine administration route, which may to some extent influence the profile of side effects associated with adjuvanted vaccine. Preventive vaccines are administered to healthy individuals including infants and children, placing special emphasis on their safety during the benefit risk ratio assessment. On the contrary, for therapeutic vaccines, an additional level of toxicity may be acceptable if the vaccine provides substantial benefit. It is thus extremely difficult to prepare pre-clinical safety regulatory guidelines that would apply to all vaccine candidates. Because the MoA of vaccine adjuvants has remained incompletely understood, potential side effects are hard to predict when an adjuvant is administered as part of a vaccine formulation, and unraveling their MoA would help in addressing safety

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considerations. Thus, as with all biological products, nonclinical safety programs for vaccines are product-specific, with adjuvants being considered an integral part of a specific vaccine formulation and unable to be licensed on their own. Hence, in addition to preclinical studies on the adjuvant itself, each new vaccineadjuvant combination will require appropriate toxicity screens and immunogenicity evaluation using the appropriate vaccination administration route before clinical trials can begin. There is a critical need for development of carefully designed in vivo models for vaccine safety analysis. Significant differences between mouse and human immunology, in both the innate and adaptive arms, were previously outlined [10e12]. Thus, although animal species cannot be bypassed for acquiring data supporting the product’s safety and efficacy, preclinical studies in animal models should be interpreted with caution. In fact, it has already been shown that a promising formulation in preclinical studies may fail in clinical trials [13]. Therefore, predictive immunological parameters and toxicology measured in preclinical studies for one given adjuvanted vaccine is dependent on vaccine formulation and may not be applicable across species or across different adjuvanted vaccines. Moreover, the risks of rare but serious AEs associated with vaccines that may occur in certain at risk populations are often nearly impossible to detect in preclinical studies and require longer follow-up of subjects from clinical studies. Therefore, one focus of scientific research is to develop new technologies to identify and validate new biomarkers of adjuvant-induced toxicity to improve preclinical safety evaluations of products containing novel adjuvants. Actually, as presented by Marina Zaitseva (Section 3.1), valuable additional data may be obtained by the use of human cell lines in vitro to detect compounds with potential in vivo toxicity. Alternative reproducible and predictive in vitro methods have been developed and are now available to complement in vivo animal studies (reviewed in Ref. [14]). 3.1. Alternative in vitro assays to predict safety/reactogenicity in humans Marina Zaitseva (Center for Biologics Evaluation and Research, FDA, Bethesda, USA) described in vitro assays based on a human monocytoid cell line, Mono Mac 6 (MM6) or human primary monocytes (Mo) [15] that could predict in vivo adjuvant toxicity and help decipher the underlying mechanisms. Pro-inflammatory cytokines (IL-1b, TNF-a, IL-6 and IL-8) and prostaglandin E2 (PGE2) secretion were used as readout for MM6 cell activation. An in vitro safety threshold of cytokine production was established by reference to 0.5 EU/ml of standard for endotoxin (an amount shown to induce a 0.5  C increase in rabbit body temperature). A comparative study of several TLR agonists (FSL-1, Pam3CSK4, flagellin, and R848) and adjuvants with established clinical profiles (Alum, MF59, Poly I:C, or MPL) demonstrated that only some TLR agonists (Pam3CSK4, FSL-1, and flagellin) induced pro-inflammatory cytokines and PGE2 above the safety threshold in MM6 cells [15]. Furthermore, in vivo pyrogenicity assessment in rabbits confirmed TLR agonist’s in vitro predicted toxicity. Indeed, PGE2 up-regulation in the plasma preceded a rise in body temperature (within 6e8 h), while CRP up-regulation in sera occurred only at 24 h [15]. Notably, following TLR stimulation in vitro, a coordinated secretion of IL-1b and PGE2 was observed, suggesting a role for the IL-1b/IL1R pathway for regulation of the systemic production of PGE2. In contrast to other cytokine activation, IL-1b requires processing from an inactive precursor by the cysteine protease caspase-1 [16]. Therefore, to investigate whether PGE2 production is dependent on the IL-1b/IL1R pathway, Mo and differentiated macrophages were cultured in vitro with a TLR-4 agonist,

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lipopolysaccharide (LPS), in the presence or absence of a caspase-1 inhibitor, ZVAD. Although ZVAD inhibited IL-1b production in both cell types, PGE2 production, as well as mRNA of two key enzymes involved in the biosynthesis of PGE2 [COX-2 and microsomal PGE synthase-1 (mPGES1)], were only partially reduced in LPS-activated monocytes. However, human recombinant IL-1b only induced PGE2, COX2 and mPGES1 proteins in LPS-primed monocytes. Therefore, additional studies were conducted to further delineate the pathway elicited by LPS. Indeed, TLR4 can elicit an inflammatory response through the adaptor molecule MyD88 or through a MyD88-independent pathway that involves alternative adaptor molecules, TRIF/TRAM, and the transcription factor IRF3 [17]. siRNA-directed inhibition of IRF3, TRIF and TRAM confirmed the role of a MyD88-independent pathway in IL-1b-induced PGE2 production in LPS-primed monocytes. Therefore, preclinical evaluation of novel adjuvants can be enhanced by a battery of human cell based assays, which are calibrated to predict in vivo reactogenicity. Moreover, these assays identified diverse PGE2 activation pathways that may translate into enhanced reactogenicity and fever in vivo, depending on the specific adjuvant product. 4. Lessons from animal models 4.1. Lessons from the veterinary field As reviewed by Bernard Charley (INRA, Jouy-en-Josas, France), the extensive use of vaccines in the veterinary field can significantly contribute to human vaccine development. Veterinary vaccines are at the forefront of the testing and commercialization of vaccines based on innovative technologies (such as recombinant subunit vaccines, genetically engineered vaccines, poxvirus vectors and DNA vaccines) [18,19]. A large number of vaccines are routinely used every year to improve the health and welfare of domestic animals, increase production of livestock and prevent animal to human transmission of zoonotic agents. They cover a wide range of vaccines formulations and adjuvants, while only a limited number of adjuvants have been approved by regulatory authorities for human use. Moreover, by using outbred populations, veterinary vaccines may better reflect individual genetic and environmental components that drive variation in immune response as compared to inbred mice contained in animal facilities, yet commonly used for preclinical evaluation of potential vaccine candidates. Another advantage of large animal models is that it is often possible to use the correct route of vaccine administration, therefore allowing a more relevant assessment of vaccine effectiveness and identification of correlates of protection [20]. Hence, veterinary vaccines may provide important information for human vaccination strategies, particularly on adjuvant efficacy, safety, potential toxicity and side effects. In addition, numerous studies are conducted in relevant target species to evaluate new vaccination strategies. For instance, in vitro and in vivo investigations for further development of intradermal vaccination methodology, particularly needle free strategies for cutaneous vaccination, are actively conducted in pigs [21]. The physiology of the skin is very similar between humans and pigs [22] and recent studies identified a potent synthetic immunoadjuvant compound that could be used for intradermal vaccine delivery using coated microneedle patches [21]. In fact, a needle-less device is already being commercialized for cutaneous vaccination on pig farms and has been shown to induce strong humoral and cellular immune responses comparable to those obtained by the intramuscular vaccination [23]. Furthermore, the adjuvant properties of several TLR ligands and cytokines are extensively studied in a number of different animal species and infection models [20,24,25].

Therefore, we can learn from the vaccination of domestic animals by linking the results obtained in mouse models, which are not always as reliable as preclinical models for human disease, to those obtained in domestic animal models. Max Bastian (Paul-Ehrlich-Institut, Langen, Germany) discussed an example of retrospective analysis of adverse reactions in bovines; particularly, the investigations into the cause of PregSureÒ Bovine Virus Diarrhea (BVD)-induced bovine neonatal pancytopenia (BNP) in newborn calves. BNP is a hemorrhagic disorder characterized by an almost complete aplasia of the red bone marrow, severe bleeding and high lethality. The disease was first reported in 2007 in Germany and neighboring countries and followed by an unexpected increase in the number of cases in some, but not all, neighboring states. The syndrome is induced in newborn calves after ingestion of colostrum, the first milk that supplies the calf with maternal antibodies. In fact, epidemiological and pharmacovigilance investigations revealed that the farms affected with BNP regularly performed vaccinations against BVD using one particular vaccination regimen [26] with one specific vaccine, PregSureÒBVD. Therefore, a study was conducted to identify the role of PregSureÒBVD in the pathogenesis of BNP [27]. Interestingly, the analysis of BNP dam sera revealed a causative role for vaccine-induced maternal alloantibodies [27]. It was demonstrated that PregSureÒBVD induces alloreactive antibodies against cell surface molecules on both the cell line used for virus production and on bovine lymphocytes, due to bioprocess related impurities originating from the bovine kidney cell line used for propagation of the virus [27,28]. Although all inactivated BVD vaccines have the potential to induce alloreactivity because they all contain a similar amount of residual bovine antigens originating from the manufacturing process, a comparative study using PregSureÒBVD [containing Immune stimulating complexes (ISCOM)like structures] and other commercial BVD vaccines (generally alum-adjuvanted) confirmed that alloreactivity was only observed in PregSureÒBVD immunized animals [27]. The comparative vaccines contained a similar amount of antigen, hence suggesting that the difference was due to the adjuvantation. Therefore, BNP is a vaccine-induced feto-maternal incompatibility syndrome caused by the combination of significant amounts of bioprocess impurities with a powerful adjuvant system. The compelling evidence for the causative role of PregSureÒBVD in BNP pathogenesis led to the withdrawal of the product from the market in August 2011. 4.2. How do animal models and humans respond to different routes of administration? Behazine Combadière (Université Pierre & Marie Curie, Paris, France) underlined that the administration route is a critical factor for successful immunization. Intradermal (ID) and transcutaneous (TC) vaccine administration techniques are currently regaining popularity as alternative routes to intramuscular (IM) or subcutaneous (SC) administrations. Indeed, different antigen presenting cells (APCs) [i.e. epidermal Langerhans cells (LCs), dermal dendritic cells (DDCs) and dermal macrophages] are targeted across the successive cutaneous layers (i.e. epidermis, dermis or hypodermis) [29,30] and the nature and quality of the immune response are differentially regulated by skin APCs [29,31e33]. A comparative study of different skin immunization routes (TC, ID needle injection and SC) in mice revealed that the route used for primary immunization strongly affects cellular and humoral immune responses [34]. Indeed, using Poly-Lactic (PLA) based nanoparticles coated with HIV-1 p24 (HIV-1 p24 PLA), the ID route induced both HIV-1 p24 specific CD8 T cells and IgG responses, whereas the SC route failed to elicit antigen-specific CD8 T cells and

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only generated specific HIV-1 p24 IgG responses [34]. Concurrent with previous findings in humans using trivalent influenza vaccine [35,36], in addition to dose sparing benefit (generally 1/5th of the dose used SC), the ID route induces higher levels of neutralizing antibodies and specific CD8 T cells compared to IM or SC routes [37e40]. The TC route of immunization through empty hair follicular ducts (by tape-stripping) favored the generation of cytotoxic CD8 T cells in the absence of IgG induction [34]. Increased CD8 T cell responses were induced in humans following TC application of influenza vaccine compared to IM immunization [41]. Remarkably, tissue-specific localization of the immune responses can be influenced by the different skin immunization routes. Substantial induction of antigen specific mucosal IgA was detected in the vaginal epithelium after TC and ID routes but not the SC route of immunization [34], an observation of great interest for improving defenses at the mucosa, the primary portal of entry for HIV infection. A recent study highlighted the involvement of epidermal LCs in particulate vaccine shuttling from the dermis to draining lymphoid tissues following dermal inoculation of Modified Vaccinia Ankara (MVA) vector [42]. Epidermal LCs were indispensable for the induction of MVA-specific CD8 T cells but not MVA-specific CD4 T cells and specific neutralizing antibodies [42]. Interestingly, additional in vivo experiments identified unexpected sources of newly primed polyclonal virus-specific CD8þ T cells in the bone marrow (BM) following ID injection of MVA [43]. These bone marrow primed CD8þ T cells exhibited a differential effector and gene expression profile compared to lymph node-primed CD8þ T cell populations. Notably, neutrophils were identified as playing a crucial role in carrying viruses from the dermis to the BM, where an interaction with resident myeloid APC resulted in an alternative source of primed CD8þ T cells [43]. Indeed, virus specific CD8þ T cell responses were lost in the BM following neutrophil depletion or by blocking neutrophil egress by using Ccr1/ mice [43]. Therefore, new vaccination strategies should consider redirecting the immune responses by targeting differential skin immunization routes that would mobilize precise arms of the immune response.

5. What can be expected from in vitro assays and animal models for prediction of adjuvant performance in humans? This question was considered by a panel made up of representatives from industry, academia and regulatory authorities. It was suggested that, for reactogenicity measurements, in vitro models may be a good starting point. Indeed, as reported by Marina Zaitseva (Center for Biologics Evaluation and Research, FDA, Bethesda, USA), alternative in vitro methods using human cell lines are now available to complement existing evaluations of vaccine safety and are in keeping with the policy to replace, reduce and refine (3Rs principle) the use of animals. However, these new approaches are not yet validated and need to be standardized by vaccine manufacturers for non-clinical vaccine studies. With reference to the use of “in vitro” testing for cosmetics the validation of such alternative methods for preclinical evaluation of vaccines is challenging due to the mixed effects of differential skin immunization routes, and the intricacy of adjuvant properties on the immune system, both locally and systemically. It was suggested that the induction of immunomodulatory cytokines may be evaluated by both tissue explants and cell lines. One major limitation of in vitro preclinical studies is the difficulty to recreate the cellular microenvironment, cellecell interactions and systemic process. In vitro methods do not always predict the entire immunomodulatory activity of an adjuvant in vivo and should be complemented by in vivo studies in appropriate animal models (if available).

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The panel acknowledged that complete understanding of the MoA of novel adjuvants may improve safety evaluation in preclinical studies. Animal models cannot yet be bypassed, though the most appropriate rather than the most convenient animal models should be selected. Indeed, mice are often the animal species of choice, very cost-effective, although they may not replicate the natural route of infection and human infections. In addition to significant biological differences between mice and humans [10e 12], housing conditions of laboratory mice represent another critical factor. Experience from vaccines in large animals in the veterinary field, which largely use the appropriate route of administration, may provide valuable information regarding risks/ benefits assessment of a specific adjuvanted vaccine. A major bottleneck in vaccine development is the lack of efficient translation of vaccine research into clinical application. The relevance of preclinical studies in animal models was debated beyond the fact they are recommended by regulatory agencies. It was even suggested that preclinical evaluations in animal models could be replaced by a phase 0 trial directly conducted in humans to help select the FIH dose for phase 1 clinical trials. However, the panel promptly acknowledged that it was inconceivable that a new vaccine in general and adjuvanted formulation could move towards clinical trials without preclinical animal safety and toxicology studies. Indeed, although toxicology studies in the vaccine industry are considered more “observational” than in the drug industry, the development of vaccine candidates has been in rare instances previously halted based on toxicity studies in rabbits. It was also suggested that preclinical vaccine evaluation may be supplemented by pharmacokinetic and biodistribution studies, currently missing. Hana Golding (Center for Biologics Evaluation and Research, FDA, Division of Viral Products, Bethesda, USA), pointed out that preclinical studies are important to decipher the MoA of novel adjuvants, to measure changes in both innate and adaptive immunological parameters, and to evaluate toxicology, thus broadening the scope of the toolbox for vaccine assessment for further development of safer vaccine formulations. Moreover, vaccine safety assessment in clinical trials and post-licensure using epidemiological studies are an essential part of ongoing monitoring of vaccines. One recommendation from regulatory agencies would be to establish a more comprehensive post-marketing surveillance, which would help in identifying high-risk subjects for an AE. 6. New technologies to predict efficacy and safety of new adjuvants/adjuvanted vaccines Recent development of powerful new technologies and computational approaches are actively contributing to the design and testing of novel vaccine formulations, and to the analysis of the complexity of the immune response in humans. Applying these technologies can help in identifying predictive safety and immunogenicity markers/biomarkers for new formulated adjuvanted vaccines and further delineating adjuvants’ MoA. 6.1. Systems biology to predict immunogenicity and safety of adjuvants The use of systems biology approaches for a comprehensive understanding of the human immune system and its regulation after vaccination was discussed by Gerlinde Obermoser (Baylor Institute for Immunology Research, Dallas, Texas, USA). Systems biology is an interdisciplinary approach that integrates, through computational analyses, high-throughput datasets from different biomic platforms (genomics, transcriptomics, proteomics and metabolomics), along with immunological and clinical parameters [44,45].

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Gerlinde Obermoser reported the results of a comparative study of the molecular and cellular immune responses to influenza (2009e2010 seasonal influenza vaccine, FluzoneÒ) and pneumococcal (23-valent pneumococcal vaccine, Pneumovax23Ò) vaccines in young, healthy, adult volunteers using systems approaches and high-throughput profiling techniques [46]. Briefly, whole blood profiling for each vaccine was generated at pre-vaccination, along with early and late post-vaccination time points (days 7, 0, 1, 3, 7, 10, 14, 21 and 28). Remarkably, transcriptional activity was detected as early as 24 h after intramuscular administration for both seasonal influenza and pneumococcal vaccines [46]. Using a genemodule analysis approach [47], the influenza vaccine was shown to induce an interferon (IFN)-inducible transcriptional signature at day 1 after vaccination, whereas the pneumococcal vaccine led to an increase of myeloid- and inflammation-related gene activity [46], thus suggesting distinct immune response pathways to immune protection. These observations were consistent with previous studies identifying IFN-related gene signature with influenza vaccine in the blood on day 1 [48] or on day 3 in purified blood mononuclear cells [49]. Moreover, molecular signature analysis of sorted cell subsets from peripheral blood mononuclear cells (PBMC) identified neutrophils and monocytes as the main source of the day 1 influenza vaccine interferon signature in the blood [46]. These results are in line with a recent study in patients with tuberculosis showing neutrophil- and monocyte-driven blood interferon signature correlating with disease severity [50]. As previously reported [49], a prominent plasmablast response was induced on day 7 by both influenza and pneumococcal vaccines, consistent with the generation of adaptive immune responses, confirmed by serum antibody responses at day 28 [46]. Of practical importance, plasmablast response could be measured in microarray profiles generated from finger pricks, making this approach attractive to studies where the amount of sample is limited, for example in very young children or critically ill patients. Finally, to extend the value of data generated in this study, novel web applications were developed which could serve as a model for future scientific publishing and data sharing. In fact, the study features interactive figures (http://www.interactivefigures.com/ dm3/vaccine-paper/figures-landing.gsp) that allow dynamic exploration of the data by the broader scientific community. Readers can interact with and customize the article’s figures by adding variables or adjusting parameters, based on their own research interests. By using this technology, human clinical trials can now be viewed as an integral part of the research continuum between discovery and clinical testing. This concept was further explored by Helder Nakaya (Emory University, Atlanta, GA, USA) who presented the use of systems biology approaches to help predict the immunogenicity and efficacy of vaccines. By using this approach, early gene signatures for predicting immune responses in humans vaccinated with the yellow fever vaccine YF-17D were identified [51]. Another example of predictive immune profiling has been demonstrated for influenza vaccines in a study of young healthy adults (18e50 years old) vaccinated with either intranasal administration of Live Attenuated Influenza Vaccine (LAIV), or intramuscular injection of Trivalent Inactivated Vaccine (TIV) [49]. This comparative study identified TIV as a higher inducer of antibody titers and plasmablasts than LAIV [49]. Distinct transcriptional signatures were identified in the blood of LAIV and TIV recipients [49]. For instance, LAIV induced higher expression of type I IFN signature genes, a common feature of the two live attenuated vaccines (LAIV [49] and YF-17D [51]), not observed after vaccination with TIV. Subsequent to these observations, two experimental approaches (molecular signatures of sorted cell subsets and meta-analysis of cell-type-specific signatures) assessed the contributions of various

PBMC subsets to the overall signature in peripheral blood cells. To avoid the considerable challenge of isolating and identifying the genomic signatures of individual subsets in the PBMC pool, a metaanalysis of cell type-specific gene-expression signatures from publicly available microarray studies was conducted and proposed as an alternative solution for deconvoluting gene-expression profiles. Consistent with molecular signatures of sorted cell subsets, metaanalysis confirmed that TIV differentially expressed genes were enriched for genes with high expression in B cells [49]. An alternative analysis, called the DAMIP (discriminant analysis via mixed integer programming) model was used to further identify predictors of TIV vaccine immunogenicity. By grouping subjects as ‘good responders’ and ‘poor responders’ based on their plasma hemagglutination-inhibition (HAI) antibody titers, early gene signatures could be used to predict with high accuracy the immunogenicity of the inactivated vaccine at day 28 in the other cohorts [49]. Notably, in subjects vaccinated with TIV, early expression levels of CaMKIV (a calmodulin-dependent protein kinase involved in neural functions, stem cell maintenance and T cell development) at day 3 following vaccination were shown to inversely correlate with HAI titers at day 28, an observation further validated using CaMKIVdeficient mice [49]. Finally, the identification of genes correlating with HAI titers also showed enrichment for genes such as TLR5 [49], suggesting that adjuvants targeting TLR5 may improve humoral responses to influenza vaccination. In fact, it has been shown that a candidate vaccine against influenza composed of a recombinant fusion protein linking influenza antigens to the TLR5 ligand flagellin may induce potent immunogenicity in mice [52] and humans [53,54]. Preliminary studies using TLR5 knock-out mice immunized with TIV showed impaired antibody responses as compared to wild-type immunized mice, thus endorsing a potential role for TLR5 in humoral responses. On the other hand, strong TLR5 agonists may lead to excessive reactogenicity in vivo [14,52,53], emphasizing the need to balance the benefit and risk of novel adjuvants and to develop tools to select the optimal dose of antigen and adjuvant, and the best formulation. In conclusion, these studies highlight how systems vaccinology has led to the discovery of molecular signatures that predict the immunogenicity of influenza vaccine and how it may help to better understand the MoA of vaccine-induced immune responses. Hence, extracting knowledge from these high-throughput techniques requires interdisciplinary tools and socio-scientific partnership between basic and translational scientists, computational biologists, clinicians, statisticians, industry, regulatory authorities, and funders. 6.2. Human-T-cell responses as predictors of effective responses Wendy Fantl (Stanford University, Stanford, California, USA) presented a recently developed technology for high-dimensional protein detection on a single-cell basis. Mass cytometry, also known as Cytometry Time Of Flight (CyTOF) is an adaptation of flow cytometry in which fluorophores are replaced by stable transition element isotopes such that atomic mass spectrometry, rather than light is the read-out. Such stable isotopes, found at low levels or absent in biological systems, are tagged to antibodies by means of a metal-chelating polymer that can be conjugated to each antibody. Currently, there are 40 available stable isotopes that can be tagged to different antibodies. Panels of antibodies can simultaneously measure surface markers, to delineate specific cell subsets, and intracellular signaling proteins with exquisite resolution between mass channels, and without cross-talk between signals (a key attribute of this technology) [55e57]. Briefly, after incubation with a panel of metal-tagged antibodies, samples are introduced into the

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mass cytometer where they are nebulized into single-cell droplets that pass through an inductively-coupled argon plasma. The resultant charged ion-cloud for each cell permits an elemental mass spectrum to be acquired for each cell [55]. The integrated elemental reporter signals for each cell can then be analyzed by using traditional flow cytometry methods, along with advanced bioinformatic tools. One algorithm, called spanning-tree progression analysis of density normalized events (SPADE) uses an unsupervised clustering algorithm to organize cells into hierarchies of related phenotypes. The cell clusters are linked into “like nearest like” to generate a minimum-spanning tree that, in addition to depicting the relatedness of cells to one another, allows a comparison of proteomic function in response to stimuli between cell clusters [58,59]. Recent studies illustrated the power of mass cytometry/SPADE by comprehensively profiling cells from healthy human bone marrow, by using two complementary 31-antibody panels. Each panel was comprised of 13-antibody surface antibody “anchor” which was occupied further by either 18 antibodies against surface markers (immune-phenotypic panel) or by 18 antibodies against intracellular signaling epitopes molecules (functional panel) [55]. The combination of the two panels revealed the hematopoietic hierarchy as well as changes in signaling responses within each cell, after exposure of samples to immune modulators and small molecule kinase inhibitors. An extension of this work incorporated markers denoting all phases of the cell cycle [59]. In both these studies hematopoiesis was revealed to be a continuum of cell immune-phenotypes. Another study used a mass cytometry platform to survey the human CD8þ cytotoxic T-cell compartment [60]. Using tetramers representing specific viral epitopes for cytomegalovirus, Epsteine Barr virus and influenza virus, it was shown that T-cells from each specificity expressed unique features [60,61]. Therefore, by adapting peptide MHC tetramer technology to mass cytometry, a detailed picture of antigen specific CD8þ T-cell diversity for the induction of intracellular cytokines, cell surface markers and cytotoxic granule components was identified [60]. The level of detail gained from single cell multi-dimensional mass cytometric analysis might provide a greater understanding of the MoA of novel vaccines/adjuvants. Combining this technology with other “omic”-based approaches during vaccine development and post-marketing surveillance, holds great promise for identifying biomarkers that could predict efficacy and safety. The expansion of specific CD4þ T cells may help predict vaccine immunogenicity in humans as presented by Flora Castellino (Novartis, Sienna, Italy). The poor predictive value of animal models for safety and immunogenicity in humans has prompted a concerted effort to define new early biomarkers predictive of vaccine immunogenicity in the blood of vaccinated individuals. It was previously demonstrated that a prime-boost strategy with MF59adjuvanted vaccines induced expansion of memory B cells, resulting in neutralizing antibodies to diverse wild-type H5N1 viruses [62]. Subsequent analysis revealed that H5 CD4þ T cell frequency after the first dose (day 21) was predictive of rise and maintenance of neutralizing antibody titers at the third dose (day 223) and 6 months later (day 382), respectively. In fact, a new CD4 helper T cell has recently emerged, termed TFH cell, found in the B cell follicles of secondary lymphoid organs and provides help to B cells to differentiate into plasma cells and memory B cells [63]. Distinguishing features of TFH cells are the expression of CXCR5, PD-1, IL-21 and ICOS, among other molecules [63]. Moreover, it was recently demonstrated that counterparts of TFH cells could be detected in blood 7 days after influenza vaccination, and provide help to memory B cells to differentiate into plasma cells [64]. Likewise, one vaccination was shown to induce the expansion of influenza specific IL-21þ ICOS1þ but CXCR5 CD4þ T cells in blood and their

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frequency is associated with functional antibodies. Moreover, circulating ICOS1þ CXCR5 CD4þ T cells were capable of helping influenza-specific B cell differentiation, in vitro, into antibody secreting cells. Therefore, it is suggested that the expansion of antigen specific IL-21þ ICOS1þ CXCR5 CD4þ T cells in peripheral blood is an early predictor of the ability of a vaccine to prime the immune system, resulting in sustained Ig titers after vaccination, and could become a useful parameter in testing vaccine immunogenicity in humans. Further analysis on the impact of age, different antigens or adjuvants on the expression of IL-21þ ICOS1þ CXCR5 CD4þ T cells in the blood of vaccinated individuals is warranted. 6.3. Dissecting the B cell repertoire to determine the shape of the immune response driven by adjuvant/delivery systems As reported by Surender Khurana (CBER/FDA, Bethesda, Maryland, USA), current efforts to develop effective pandemic influenza virus vaccines, including H5N1, are hampered by a lack of knowledge in immune correlates of protection in humans. Therefore, to better understand protective immunity against avian influenza, the whole-genome-fragment phage display libraries (GFPDL) technique was adapted to decipher the complete antibody epitope repertoire following natural exposure or influenza vaccination. This approach uses genetically modified viruses (phages) to create a library of protein fragments that span all the open reading frames of H5N1 A/Vietnam/1203/2004. Mixing the GFPDL-protein fragments with sera from convalescent individuals who recovered from H5N1 virus infection unraveled small and large conformation dependent epitopes of several H5N1 proteins that assisted in development of new serodiagnostic test for H5N1 exposure [65]. In evaluating vaccine responses, adding adjuvants such as MF59 was required to improve immune responses and for cross-protection against drifted avian H5N1 influenza viruses. The effect of MF59 on the immunogenicity of the H5N1 inactivated vaccine was analyzed in sera of vaccinated adults using GFPDL [66]. Furthermore, analysis of polyclonal antibody affinity in human sera was conducted using surface plasmon resonance (SPR) [66]. It was shown that MF59 adjuvantation was able to expand the anti-hemagglutinin (HA) antibody repertoire, spreading the targeted epitopes from predominantly HA2 sequences to sequences in HA1 (receptor binding domain), which contains most of the neutralizing epitopes, and neuraminidase (NA) (sialic acid binding site) [66]. Moreover, the shift in the antibody repertoire correlated with broadening of cross-clade neutralization [66]. Likewise, the added value of MF59 on the antibody responses induced by swine-origin influenza virus (pH1N1) or avian-derived H5N1 vaccines was evaluated in various age groups and showed enhanced functional antibody responses to HA-based vaccines by improving both epitope breadth and binding affinity [67]. Another approach to improve the breadth and affinity of antibody responses to H5N1 monovalent inactivated vaccine (MIV) in adults was demonstrated using H5 DNA priming followed by H5N1 MIV boost over the homologous MIVeMIV prime/boost protocol [68]. H5 DNA vaccine consists of a single closed circular plasmid DNA macromolecule expressing influenza (A/Indonesia/5/2005) HA sequence, derived from a human isolate [68]. In humans, it was previously shown that H5 DNA priming could improve the antibody response to a boost with inactivated influenza vaccine given 24 weeks later [69]. Using GFPDL and SPR technologies, it was shown that H5 DNA priming expanded H5-specific targeted epitopes (in HA1) and enhanced antibody avidity to the HA1 domain (not HA2), in a prime/boost interval-dependent manner [68]. In fact, the shortest interval required for significant changes in the quality of the polyclonal antibody responses was 12 weeks long [68,70], and the interval length strongly correlated with improved neutralization of

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homologous and heterologous H5N1 strains [68]. Hence, the use of GFPDL and SPR led to new insights into the repertoire and quality of the human polyclonal antibody response following infection and/or vaccinations against the avian influenza highly pathogenic H5N1 virus with pandemic potential. Therefore, these technologies may have significant implications for future vaccine design: deciphering the quality of immune responses induced by novel candidate vaccine formulations and adjuvants. Gary Nabel (Sanofi, Cambridge, Massachusetts, USA) discussed another prime/boost vaccination strategy capable to induce broadly neutralizing influenza antibodies. Gene-based priming with plasmid DNA encoding H1N1 influenza HA and boosting with seasonal trivalent vaccine or replication-defective adenovirus 5 (rAd5) vector encoding HA could broaden the range of neutralizing antibodies against H1N1 strains recovered between 1934 and 2007 [71]. Remarkably, these antibodies were directed to the conserved stem region of HA protein [71], while the majority of current vaccines elicit influenza antibodies directed to the globular head region of viral surface glycoprotein HA, which undergoes considerable antigenic drift to evade the human immune system. Thus, ways to elicit antibodies targeting the highly conserved HA stem epitope and to develop universal influenza vaccines were increasingly explored. Two specific hydrophobic residues in the CDRH2 region of broadly neutralizing MAb were identified as determinants for engagement of the highly conserved stem region of influenza HA [72]. Recently, a novel vaccine platform, the ferritin nanoparticles, was introduced as a powerful technology for delivery of influenza vaccines [73]. Ferritin, a ubiquitous iron storage protein, selfassembles into spherical nanoparticles which can serve as a scaffold to express influenza HA [73]. Viral HA was inserted at the interface of adjacent subunits so that it spontaneously assembled and mimicked trimeric HA spike on its surface [73]. Remarkably, HA-nanoparticles enhanced the magnitude and breadth of broadly neutralizing antibody responses, more than 10 fold higher than a matched inactivated vaccine (TIV) [73]. For instance, antibodies elicited by a 1999 HA-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge [73]. In fact, two types of broadly neutralizing antibodies directed to two independent highly conserved epitopes were elicited: one directed to the highly conserved HA stem (neutralizes virus by inhibiting membrane fusion) and a second proximal to the conserved receptor binding site on the HA head (inhibits viral entry) [73]. It was shown that HA-nanoparticles could be associated with an adjuvant, such as MF59, to induce broad and potent immune responses [73]. Therefore, these self-assembling, easy to manufacture, synthetic nanoparticle vaccines represent a promising new approach to future vaccine development against emerging influenza viruses and other pathogens. 6.4. What can we expect from these new technologies? This question was considered by a panel made up of representatives from industry, academia and regulatory authorities. Thanks to these new technologies, tremendous progress has been made in the field of vaccine immunology and very powerful tools have been created for studying human immune responses and vaccine mechanisms, thus narrowing the gap between basic research and clinical evaluation. These exploratory tools should help reduce the risk of failure at an earlier stage of vaccine development, a beneficial feature, while progressing to later stages is becoming more and more resource-consuming. Hitherto, these assays have been applied retrospectively to refine the understanding of the vaccine MoA and to potentially identify immune correlates of protection.

Concerted efforts are needed to verify reproducibility of the assays and to standardize them before considering these novel approaches for non-clinical and clinical studies, so as to complement current assessment strategies for safety and effectiveness. Yet, there may be risks involved when converting these tools into regulatory requirements. For instance, because of the significant cost, small companies may not have enough resources to use these technologies on a regular basis during preclinical and clinical evaluations. Furthermore, as these techniques generate a flood of increasingly complex data, a public-private partnership should be encouraged to optimize data collection and analysis for identifying compound mode of action. 7. Regulatory challenges related to licensure of adjuvanted vaccines With the development of novel vaccines, the design of vaccine products has evolved, combining novel adjuvants, new delivery systems and alternate routes of administration. The introduction of an adjuvant into a vaccine presents challenges for regulatory evaluation. In addition, at the present time, vaccine regulatory procedures across the USA and Europe are not harmonized; there are discrepancies between requested regulatory toxicology and risk assessment processes and no mutual recognition of licensing. Whereas adjuvants are considered as an active component of an adjuvanted vaccine from an immunological viewpoint, the question has been raised as to whether adjuvants should be considered as active ingredients, or as excipients, with ensuing differential impact on regulatory requirements. Marion Gruber (FDA, Rockville, Maryland, USA) discussed the regulatory pathways supporting the development and approval of vaccines formulated with novel adjuvants in the United States. There is a rigorous review of laboratory and clinical data to ensure vaccine safety, efficacy, purity and potency prior to marketing [74]. She drew attention to the fact that under the US regulatory considerations, adjuvants are considered as inactive ingredients, as described in the Code of Federal Regulations (CFR). Based on this classification, adjuvants are not licensed on their own but rather as a constituent material of vaccine formulations (21CFR 610.15). For the presence of an adjuvant in a vaccine to be accepted, manufacturers must provide a strong rationale for its incorporation in the vaccine formulation, as well as data regarding the safety of the vaccine-adjuvant formulation. Justification of the adjuvant may be obtained from preclinical studies (“in vitro” assays, animal models) and/or from phase I and II studies designed to compare vaccine formulation with and without adjuvant. Demonstrating the “added benefit” in clinical phase III studies comparing adjuvanted vs. unadjuvanted vaccines is not a regulatory requirement, although it may be requested on a case-by-case basis, e.g., if a manufacturer wants to make a claim of superiority for the adjuvanted vaccine. The safety of the adjuvanted vaccine must be demonstrated in pre-licensure safety studies whereby it is not necessary to compare the adjuvanted vaccine to the unadjuvanted vaccine. To identify potential late onset AEs, including autoimmune symptoms, a 12month follow-up of study participants is usually requested following the last vaccination. The size of the pre-licensure safety study would also be influenced by the clinical experience derived from studies using the same adjuvant and formulated with different vaccine antigens. Pieter Neels (EMA, United Kingdom) noted that whilst adjuvants are highly heterogeneous in their mechanisms of action, they are generally classified as excipients for regulatory purposes in the European Union. Adjuvants are defined as components that increase specific immune responses to an antigen and should thus be part of the reconstituted vaccine formulation that is administered

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simultaneously or concomitantly with the vaccine antigen [74]. While several guidelines for use of adjuvants are available (Guideline on clinical evaluation of new vaccines, EMEA/CHMP/ VWP/164653/2005; Guideline on adjuvants in vaccines for human use, EMEA/CHMP/VEG/134716/2004; new draft WHO guideline, upcoming) and cover almost all issues, there is still a lot of concern surrounding their safety. For instance, recent reports of narcolepsy following H1N1 vaccination led to further community resistance towards vaccination and adjuvants [75,76]. It has been suggested that vaccination with AS03 pandemic H1N1 vaccine contributed to the onset of narcolepsy during the 2009e2010 influenza pandemic [75,76]. However, the pathogenesis of narcolepsy as an immunopathology is not clear and the causal pathways between vaccination and this condition have yet to be unraveled [77]. Pieter Neels thus concluded that while such safety concerns may be justified, there exists a knowledge gap regarding such associations. Regulatory challenges and common issues were discussed by all conference participants. Regional differences in regulatory definitions of an adjuvant were broadly debated. If adjuvants were to be considered active ingredients from a regulatory perspective, clinical trials demonstrating that each active ingredient in the vaccine formulation contributes to the claimed effect would be required. Thus, this may significantly increase the size and cost of clinical trials. If considered excipients, such clinical studies would not need to be required. In fact, a valuable effect of an adjuvant is gained only when combined with a specific antigen. In addition, while investigating the contribution of the individual component of MF59 o/w emulsion, it was demonstrated that only the fully formulated MF59 emulsion is an active adjuvant [78]. As for any preventive vaccine, safety of adjuvanted vaccines is an important consideration, therefore, it was suggested that safety data should be shared among manufacturers, as this could protect clinical study participants from unnecessary harm. However, it was noted that this approach might be limited by the lack of uniformity in definition and evaluation of safety events. Other considerations included that, in addition to safety requirements, any superiority claims from manufacturers for any adjuvanted vaccines would need to be demonstrated in clinical trials to a licensed comparator. Therefore, it was suggested that a new harmonized regulatory lexicon should be considered, redefining adjuvants and, where possible, harmonized regulatory requirements with the objective of a more reasonable use of animal and material resources. New regulatory frameworks and pathways to licensure may need to be developed, e.g. pandemic influenza vaccines formulated with novel adjuvant. In conclusion, nonclinical safety evaluation of new vaccine formulations is a critical step in the overall development of vaccines prior to initiating clinical investigations and receiving marketing approval. One of the challenges for nonclinical safety assessment is to select the relevant animal model to predict adjuvant/vaccine associated risks in humans. In line with the 3Rs principle, in vitro assays should be validated to help select and predict the in vivo activity and toxicity of a novel adjuvant. Further research is needed to better understand the MoA of adjuvants and to identify potential biomarkers for the preclinical safety evaluation of a vaccine during the early stages of development, in order to improve vaccine safety assessment. Acknowledgments We would like to thank members of the IABS board, the scientific committee and the speakers for their excellent organization of the meeting. This workshop was made possible by unrestricted educational grants from CSL, Sanofi Pasteur and Novartis. We would also like to thank the U.S. Pharmacopeial Convention (USP) for the perfect venue. We would like to thank Victor Popoola for his valuable contribution for the manuscript revision.

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Appendix A. List of participants Brigitte Autran, University Pierre & Marie Curie, Paris, France; Max Bastian, Paul-Ehrlich-Institut, Langen, Germany; Steven Black, Cincinnati Children’s Hospital, Cincinnati, Ohio, USA; Flora Castellino, Novartis, Siena, Italy; Bernard Charley, Institut National de la Recherche Agronomique (INRA), Jouy-en-Josas, France; Behazine Combadière, Pierre & Marie Curie, Paris, France; Giuseppe Del Giudice, Novartis, Siena, Italy; Arnaud Didierlaurent, GlaxoSmithKline Vaccines, Rixensart, Belgium; Joe Francica, VRC/NIH, Bethesda, Maryland, USA; Bernard Fritzell, co-Chair, Human Vaccine Committee, IABS; Nathalie Garçon, GlaxoSmithKline Vaccines, Rixensart, Belgium; Hana Golding, CBER/FDA, Bethesda, Maryland, USA; Marion Gruber, FDA, Rockville, Maryland, USA; Surender Khurana, CBER/FDA, Bethesda, Maryland; David Lewis, University of Surrey, United Kingdom; Arnaud Marchant, Université Libre de Bruxelles, Belgium; Gary Nabel, CVR/NIH, Bethesda, Maryland, USA; Helder Nakaya, Emory University, Atlanta, Georgia, USA; Pieter Neels, EMA, United Kingdom; Gerlinde Obermoser, Baylor Institute for Immunology Research, Dallas, Texas, USA; Marc Pallardy, Faculté de Pharmacie Paris-Sud, France; John Pettricciani, President, IABS; Marie Joelle Frachette, Sanofi Pasteur, Lyon, France; Marina Zaitseva, CBER/FDA, Bethesda, Maryland, USA; Wendy Fantl, Stanford University, Stanford, California, USA; Betty Dodet, IABS committee, France; Abigail Charlet, IABS committee, France. References [1] Mastelic B, Ahmed S, Egan WM, Del Giudice G, Golding H, Gust I, et al. Mode of action of adjuvants: implications for vaccine safety and design. Biologicals 2010;38:594e601. [2] De Gregorio E, Tritto E, Rappuoli R. Alum adjuvanticity: unraveling a century old mystery. Eur J Immunol 2008;38:2068e71. [3] Batista-Duharte A, Lindblad EB, Oviedo-Orta E. Progress in understanding adjuvant immunotoxicity mechanisms. Toxicol Lett 2011;203:97e105. [4] Coffman RL, Sher A, Seder RA. Vaccine adjuvants: putting innate immunity to work. Immunity 2010;33:492e503. [5] Shao W, Boltz VF, Spindler JE, Kearney MF, Maldarelli F, Mellors JW, et al. Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of low-frequency drug resistance mutations in HIV-1 DNA. Retrovirology 2013;10:18. [6] Brennan FR, Dougan G. Non-clinical safety evaluation of novel vaccines and adjuvants: new products, new strategies. Vaccine 2005;23:3210e22. [7] Didierlaurent AM, Morel S, Lockman L, Giannini SL, Bisteau M, Carlsen H, et al. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity. J Immunol 2009;183:6186e97. [8] Morel S, Didierlaurent A, Bourguignon P, Delhaye S, Baras B, Jacob V, et al. Adjuvant system AS03 containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity. Vaccine 2011;29: 2461e73. [9] Mullen GE, Ellis RD, Miura K, Malkin E, Nolan C, Hay M, et al. Phase 1 trial of AMA1-C1/alhydrogel plus CPG 7909: an asexual blood-stage vaccine for Plasmodium falciparum malaria. PLoS One 2008;3:e2940. [10] Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731e8. [11] Schroder K, Irvine KM, Taylor MS, Bokil NJ, Le Cao KA, Masterman KA, et al. Conservation and divergence in toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages. Proc Natl Acad Sci U S A 2012;109:E944e53. [12] Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A 2013;110:3507e12. [13] Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA, Lockhart S, et al. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 2013;381:1021e8. [14] Mastelic B, Lewis DJ, Golding H, Gust I, Sheets R, Lambert PH. Potential use of inflammation and early immunological event biomarkers in assessing vaccine safety. Biologicals 2013;41:115e24. [15] Zaitseva M, Romantseva T, Blinova K, Beren J, Sirota L, Drane D, et al. Use of human MonoMac6 cells for development of in vitro assay predictive of adjuvant safety in vivo. Vaccine 2012;30:4859e65. [16] Tschopp J, Schroder K. NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat Rev Immunol 2010;10: 210e5.

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Please cite this article in press as: Mastelic B, et al., Predictive markers of safety and immunogenicity of adjuvanted vaccines, Biologicals (2013), http://dx.doi.org/10.1016/j.biologicals.2013.08.006

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Please cite this article in press as: Mastelic B, et al., Predictive markers of safety and immunogenicity of adjuvanted vaccines, Biologicals (2013), http://dx.doi.org/10.1016/j.biologicals.2013.08.006