Vaccine 33S (2015) B14–B20

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Vaccine journal homepage: www.elsevier.com/locate/vaccine

Review

New generation adjuvants – From empiricism to rational design Derek T. O’Hagan a , Christopher B. Fox b,∗ a b

Novartis Vaccines, Cambridge, MA, United States IDRI, Seattle, WA, United States

a r t i c l e

i n f o

Keywords: Vaccine adjuvant Alum Emulsion MPL Formulation

a b s t r a c t Adjuvants are an essential component of modern vaccine development. Despite many decades of development, only a few types of adjuvants are currently included in vaccines approved for human use. In order to better understand the reasons that development of some adjuvants succeeded while many others failed, we discuss some of the common attributes of successful first generation adjuvants. Next, we evaluate current trends in the development of second generation adjuvants, including the potential advantages of rationally designed synthetic immune potentiators appropriately formulated. Finally, we discuss desirable attributes of next generation adjuvants. Throughout, we emphasize that the importance of formulation and analytical characterization in all aspects of vaccine adjuvant development is often underappreciated. We highlight the formulation factors that must be evaluated in order to optimize interactions between vaccine antigens, immune potentiators, and particulate formulations, and the resulting effects on safety, biological activity, manufacturability, and stability. © 2015 Elsevier Ltd. All rights reserved.

1. Empirical vaccine adjuvants – the first generation particulates The first generation of adjuvants that have been widely available for many years and some of which are included in the currently licensed vaccine products, have been described by Steve Reed (in this issue). These adjuvants are essentially particulate ‘carriers’, which although are often compositionally and structurally very different, have broadly similar dimensions (Fig. 1) and closely related mechanisms of action. Adjuvants based on insoluble aluminium salts, oil in water emulsions and liposomes have been used in human vaccines for some considerable time and have enjoyed significant success as components of licensed products. In contrast some alternative particulate adjuvants described in more recent decades, e.g. ISCOMs and polymeric particles, have made more limited progress into clinical testing, and have not yet been included in vaccine products. Since only a few adjuvant technologies have succeeded, while many others have failed, we would like to use our experience and insights to highlight what might have made the difference and importantly, to try to improve success rates in the future. In thinking about why so many have failed, perhaps an

∗ Corresponding author. Tel.: +1 206 858 6027. E-mail address: [email protected] (C.B. Fox). http://dx.doi.org/10.1016/j.vaccine.2015.01.088 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

important consideration is that those which succeeded usually had an alternative medical use prior to their utilization in vaccines (Table 1). Moreover, the alternative uses often continued and expanded alongside their use in vaccines, which meant that many key technical attributes were in place, which could also be exploited for vaccines. For example, the manufacturing of formulations suitable for clinical evaluation was established, using materials that had been resourced through a supply chain which was appropriate for a medical product. Moreover, the use of these technologies (e.g. liposomes/emulsions) in vaccines could directly benefit from process or manufacturing improvements that were implemented due to alternative uses of the technologies. We believe that perhaps this point has been under-appreciated, and that it should be an important consideration as we consider the question of ‘which are the best adjuvant technologies for the future?’ The economics of vaccine development has traditionally been challenging and the market realities have made it difficult to support large and expensive manufacturing investments for new technologies. Therefore, it is attractive if within the pharmaceutical industry an alternative product opportunity outside the vaccine arena could support the development process for a new adjuvant, particularly since the vaccine industry operates substantially within the allied pharmaceutical industry. Unfortunately, this may suggest that many of the new adjuvants currently under investigation are likely to fail, like many more before them.

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Table 1 Alternative use of clinical phase adjuvants. Delivery system/adjuvant

Early medical use

Licensed for drugs

Aluminium salts Emulsions (o/w) Liposomes/virosomes Topical cream w/TLR7 agonist Microparticles (PLG) Saponins (ISCOMs, etc.)

Oral ∼1915 Intralipid TPN–1962 Artificial membranes – 1964 Genital warts, actinic keratosis, carcinoma Steroid hormones – 1980 Veterinary vaccines – 1951

Yes – oral antacids Yes – Propofol (1989) Yes – Doxil (1995) now >10 Yes – Aldara (1997) Yes – hGH (1999), sutures (1970) Yes – veterinary vaccines (1951)

2. Second generation adjuvants – exploiting synergy between the first generation and added immune potentiators The majority of second generation adjuvants currently under investigation have exploited the successes of the first generation, while adding an ‘immune potentiator’ to improve potency. The added immune potentiators are usually TLR agonists, as discussed by Steve Reed (in this issue), although alternative pattern recognition receptor (PRR) ligands are also available to potentially exploit additional or alternative pathways of innate activation, including NLR, RIG-I or STING. The addition of TLR agonists is not a new concept, since combined vaccine adjuvants have been evaluated since the 1930s, when whole bacterial cells were added to water-inoil emulsions to create Freunds’ complete adjuvant. Nevertheless, successful licensure of a product containing a purified bacterial component as a component of a second generation adjuvant was not achieved until 2005 [1]. Monophosphoryl lipid A (MPL® ), a TLR4 ligand, was the first TLR agonist included in a licensed human vaccine. Although MPL® is a natural product, there are now various synthetic TLR4 agonists available with potential advantages over the original natural product, including GLA (Reed et al., in this

issue). Moreover, besides TLR4 ligands, there are various other TLR agonists at various stages of clinical development. Overall, there is a gradual and logical shift from the use of natural products to more rationally defined and synthetically created TLR agonists and others for inclusion in second generation adjuvants. In general, we believe that the key role of formulation science has traditionally been underappreciated in adjuvant development. Moreover, it is within the context of the development of second generation adjuvants that this role becomes most crucial. Since there are a range of particulate adjuvants available, which can now be combined with a range of immune potentiators (TLR agonists and others), it is the crucial and distinctive role of formulation science to determine how best these can be optimally combined. In addition, the combination (2nd generation) adjuvants can be linked to vaccine antigens in a variety of ways including adsorption, encapsulation, conjugation, complexation, chelation, dispersion or simple co-administration. Unfortunately, the actual need for physical association between individual components needs to be determined empirically, and remains both antigen and immune potentiator dependent. Extensive studies need to be undertaken to address this question for each new adjuvant, and these need to be driven by insightful formulation science. The objective of these

Fig. 1. Electron micrographs of adjuvant formulations demonstrate the complexity and diversity of particulate structures. (a) Unstained TEM of aluminium oxyhydroxide (scale bar 2 ␮m), (b) cryo-TEM of GLA-liposomes (scale bar 200 nm), (c) cryo-TEM of GLA-SE, (d) negative stained TEM of ISCOMs (scale bar 100 nm), (e) SEM of PLG nanoparticles (scale bar 10 ␮m). (a) Reprinted from Harris et al. Micron 2012, 43:192–200, with permission from Elsevier. (b and c) Reprinted from Fox et al. [3], with permission from Elsevier. (d) Reprinted by permission from Macmillan Publishers Ltd: Sanders et al. Immunol Cell Biol 2005, 83:119–128.

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studies should be to selectively modify the adjuvant formulation to optimally exploit the innate activation signals, to ensure the development of a safe and effective vaccine. The adjuvant formulation design must also consider issues of manufacturability and stability, if it is to find a role in the ‘real’ world of practical vaccinology. Formulation scientists are needed to ensure that each vaccine component is stable in the presence of the others and is located optimally to ensure both safety, and the best immune response. Unfortunately, this key role has so far been underappreciated, but this needs to change if we are to have more success in developing new and improved adjuvants in the coming decades.

3. Antigen/immune potentiator delivery – formulation matters! Perhaps the clearest indication that formulation of immune potentiators really matters in terms of potency comes from the Phase II malaria human challenge studies of GSK. In these studies, they showed that a liposomal formulation of MPL® and a saponin adjuvant (QS21) offered better protection against parasitaemia than an emulsion formulation of the same components [2]. However, it is not necessary to perform human challenge studies to discern key differences in adjuvant formulations. Fox et al. showed significant differences in potency in a mouse model for the induction of CD4+ responses when they compared several alternative formulations of GLA with malaria antigens [3]. In this study it was the emulsion formulation (GLA-SE) that was the most potent, in comparison to a liposome or an aqueous dispersion (Fig. 1). Although this may appear to contradict the observations in man for the MPL® -containing formulations, we should emphasize that the synthetically produced GLA is different from the natural product MPL® (Reed et al.) and that the GSK formulations also contained QS21. In addition, the liposomal and emulsion formulations differed in excipient content, e.g. GSK’s emulsion was comprised of squalene and ␣-tocopherol, whereas IDRI’s emulsion consisted of squalene alone. Moreover, the antigens used and the interactions between antigen and adjuvant were also different in these studies. Nevertheless, perhaps the imminent licensure of a malaria vaccine (Mosquirix® ) containing the AS01 adjuvant formulation, comprising liposomal MPL® and QS21, represents a potential renaissance for liposomal adjuvants. Certainly liposomes have many attractive attributes, since they can entrap and deliver lipophilic immune potentiators, and they can be scaled up and developed as a clinical product. Currently liposomes are being used for alternative medical uses, since there are now >10 licensed products available, mainly as drug delivery systems. Furthermore, liposome-based antigen delivery systems known as virosomes have been employed for several years in approved vaccines, both for seasonal influenza and hepatitis A (Crucell’s Inflexal® and Epaxal® , respectively). Moreover, in comparison to other adjuvant options, liposomes are probably the most ‘benign’ of the carriers available, since non-charged phospholipids alone do not appear to significantly activate the innate system. Therefore, immune potentiators can be included in the liposomes to steer the immune response in the preferred direction, without much interference from the ‘cell membrane like’ formulation composition. However, charged lipids with immune stimulatory capabilities can also be included in liposomes if preferred, e.g. CAF01, although this is likely to have a negative impact on the tolerability profile. Nevertheless, liposomes may not be the best option if a stable single vial liquid formulation is the preferred outcome for the vaccine. In contrast, stable single vial vaccines are often possible with the more established aluminium based adjuvants. The Mosquirix® vaccine will comprise a lyophilized antigen to be reconstituted with liquid AS01.

4. Potential next generation adjuvants currently under evaluation There are a number of potential new generation adjuvant candidates that are currently under clinical investigation (Table 2), which are not based on the typical first generation approaches, including CAF01 (a cationic liposome containing a synthetic C-type lectin agonist) and a particulate construct called IC31 (combines a cationic peptide with a TLR9-activating oligonucleotide) [4,5]. Interestingly, both CAF01 and IC31 have been shown to function in part by providing a ‘depot’ mechanism, although this mechanism rather ‘fell from favor’ as an explanation of how Alum based adjuvants worked. Moreover, the overall value of having a mechanism based on an antigen depot may be questioned in light of the recent finding that depot-based adjuvants appear to trigger T cells that ‘attack’ the injection site rather than the sites where the pathogens might actually reside [6]. Other adjuvants that have been evaluated clinically include the synthetic small molecule imiquimod (TLR7 agonist), which is approved in a topical cream formulation (Aldara® ) for treatment of genital warts, actinic keratosis, and basal cell carcinoma. Interestingly, in a clinical trial of elderly participants, Aldara® applied topically immediately prior to intradermal immunization with influenza vaccine resulted in significantly higher seroconversion rates [7]. In addition, Dynavax’s HEPLISAVTM , an improved hepatitis B vaccine comprising a recombinant antigen (and containing a TLR9 agonist) has completed phase III clinical testing, although the FDA has requested additional safety testing prior to licensure. Another adjuvant technology currently under clinical evaluation is represented by ISCOMs, which are particulate structures comprising phospholipids and saponins, described originally in the 1980s (Fig. 1) [8]. Although the original technology was perhaps too reactogenic for widespread use, various refinements have been made, particularly in relation to the composition of the saponin fractions used. The latest iteration of this technology, a formulation called Matrix-MTM is currently in Phase 2 clinical testing as an adjuvant for a flu vaccine. However, if an adjuvant technology needs the inclusion of a saponin, the success of the AS01 approach might suggest that it is perhaps best to deliver the saponin in a liposome rather than an ISCOM. Moreover, as discussed earlier, the potential approval of the AS01-containing Mosquirix® vaccine may pave the way for additional improved liposomal formulations that are currently in much earlier stages of development, including the interbilayer-crosslinked multilamellar vesicles (ICMVs) [9]. Overall, it should be clear that the path to success for new adjuvants is certainly a long and challenging one, but the first and most important hurdle is that they will have to demonstrate an excellent safety profile when included in a vaccine, particularly if they are proposed for use in children. Although limited local reactions may be acceptable, a significant incidence of systemic reactions is most likely to prove problematical. Nevertheless, each vaccine product will be considered for licensure with a careful consideration of the overall risk/benefit analysis, which will weigh the adverse event and safety profile versus the ongoing incidence of target disease and disease outcomes. Hence, any vaccine with a novel adjuvant is perhaps best targeted to try to overcome a significant unmet need, to protect against an infection which is not yet under vaccine control, rather than to be a marginal improvement on an already existing vaccine.

5. Which is the best delivery system for the future? While alum, oil-in-water emulsion, and liposome-based adjuvants are already included in licensed vaccine products, perhaps the most optimal ‘delivery’ adjuvant for future use has yet to be identified. One potential approach that has seen a resurgence of

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Table 2 Current status of selected adjuvants in development for prophylactic indications. Adjuvant (examples)

Description

Development status

Aluminium salts

Insoluble particulates of hydroxide, phosphate or hydroxyphosphate sulfate salts Oil dispersed nanoemulsions (mainly squalene) stabilized with non-ionic surfactants Dispersed lipid vesicles containing TLR4 ligand and saponin Dispersed lipid vesicles including viral membrane (influenza) proteins Topical ointment of TLR7 ligand (Aldara) applied in conjunction with intradermal vaccination Biodegradable polymeric microparticles Lipid, purified saponins and cholesterol cage-like nanocomplexes Natural product TLR4 ligand (MPL) adsorbed to aluminium hydroxide Synthetic TLR4 ligand adsorbed to aluminium hydroxide Soluble TLR9 ligand (oligonucleotide) Oil-in-water nanoemulsion with TLR4 ligand (MPL) and saponin Oil-in-water nanoemulsion with synthetic TLR4 ligand (GLA) Cationic peptide complexed with TLR9 ligand (oligonucleotide) TLR5 ligand protein linked to antigen Double stranded RNA polymer analogue (PolyI:C12U) TLR3 ligand Plant-derived crystallized microparticulate polysaccharide Cationic liposome including a synthetic CLR ligand Small molecule synthetic TLR7 ligand adsorbed to aluminium hydroxide

Included in licensed products for routine childhood vaccines and many others Included in licensed products for seasonal influenza (MF59) or pandemic influenza (MF59, AS03) Phase III, submitted for licensure for malaria

Oil-in-water emulsions (MF59, AS03, etc.) Liposomes (AS01) Virosomes Topical cream w/TLR7 ligand Polymeric microparticles (PLG) Saponin complexes (ISCOM, Matrix-M) AS04 RC-529 CpG ODN (1018 ISS) AS02 GLA-SE IC31 Flagellin Poly(I:C) (Ampligen) Delta inulin (Advax) CAF01 Alum/TLR7

interest in the recent past is represented by biodegradable polymeric PLG particles (Fig. 1). These particles were first described for antigen delivery in the early 1990s, when they were proposed as controlled release vaccines to obviate the need for booster doses. However, a decade later this concept was largely considered to have failed, mainly due to the difficulties of stabilizing antigen within the particles [10]. Nevertheless, the technology re-emerged a few years ago as a potential combined delivery system for antigen and TLR agonists (TLR4 and TLR7) [11]. Although PLG particles do possess some of the key attributes that may allow them to potentially succeed as an adjuvant, the problem of degradation of antigen entrapped in the particles has not yet been overcome. This problem was ‘masked’ in the recent high profile study [16], since the antigen evaluated was a formalin fixed whole virus, which is not representative of a wider range of newer generation recombinant vaccines currently under evaluation. Unfortunately, more typical vaccine antigens have been shown on many occasions to be generally unstable when entrapped in PLG particles [10]. Nevertheless, an alternative approach has been described in which the PLG particles can be used as a carrier for adsorbed antigen, similar to Alum, but PLG has the advantage of being completely biodegradable [12]. The antigen adsorption approach can overcome the problems of antigen degradation for recombinant antigens [13] and has also been adapted to allow simultaneous delivery of TLR agonists [14]. The adjuvant potency of PLG particles was significantly enhanced through encapsulation of a TLR4 agonist, while full antigen stability was retained due to adsorption [19]. However, in the bigger picture, we should not lose sight of the fact that since PLG is a biodegradable polymer, it will always need to be a lyophilized formulation which tends to increase costs, while reducing ease of use in the field. Although a lyophilized product has potential advantages for long term vaccine stability, perhaps even at elevated temperatures, it moves away from the ideal concept of an inexpensive single vial liquid formulation that is currently offered by Alum. One question requiring further work in relation to the potential of PLG or other particles as adjuvants is ‘what is the optimal size for

Licensed products for influenza and hepatitis A Phase III for influenza Phase I Phase II for influenza Licensed products for HBV and HPV Was licensed product in Argentina for HBV Phase III, submitted for licensure for HBV Phase III Phase II Phase II Phase II Phase II Phase II Phase I Phase I

improved potency?’ Although Kasturi et al. used nanoparticles to show that PLG could deliver more than one TLR agonist, there was no comparison with the more established microparticles [11]. It has been claimed previously on various occasions that nanoparticles are more potent than microparticles, but we believe that well controlled data is often lacking to support this assertion and more work is needed [15]. Nevertheless, even if the potency is the same for micro- and nanoparticles, nanoparticles may offer additional advantages, including that they can be produced without an expensive aseptic manufacturing process that is required for microparticles, since nanoparticles can be sterile filtered [16]. Moreover, for surface adsorbed antigen, nanoparticles have higher loading capacity due to a larger surface area, which might be beneficial for the delivery of complex combination vaccines. Nevertheless, an alternative approach to avoid aseptic manufacturing is possible, since it has been shown that microparticles can be sterilized by gamma irradiation, before the antigens are adsorbed [17].

6. Which is the best kind of immune potentiator for the future? Early efforts to develop immune potentiators focused primarily on naturally derived, heterogeneous mixtures, including MPL (derived from Salmonella minnesota) and saponins, such as those found in ISCOMs (derived from Quillaja saponaria bark). Unsurprisingly, since these materials are naturally derived, well controlled, reproducible manufacture can be challenging. Consequently, newer approaches have focused on the development of pure, synthetic materials, such as the TLR4 ligand GLA (containing the hexa-acylated molecule optimal for human TLR4 engagement) and semisynthetic QS21 saponin. However, if the natural product is already established, e.g. MPL, the impact on cost or safety differences for the synthetic molecules must be considered in the course of potential product development. Thus, semisynthetic QS21 is envisioned as a potential component of a cancer immunotherapy

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product, but probably not cost effective as a standard vaccine component [18], although simplified structures may be more tractable [19]. Optimized synthetic molecules with improved potency may result in lower adjuvant doses in the clinic. For example, GLA doses in the clinic are an order of magnitude lower than MPL doses in Mosquirix® and Cervarix® , due to the enhanced potency of the purely hexa-acylated adjuvant. In addition, synthetic small molecule imidazoquinolines, which are TLR7/8 ligands, can be very cost effective to make in comparison to existing adjuvant molecules. However, typically the unformulated small molecule adjuvants do not work well in comparison to their larger molecular weight counterparts, since they are prone to diffuse away from the injection site, which can also result in systemic toxicity. Nevertheless, if new formulation concepts can be developed to better retain the small molecules at the injection site, then they may represent an excellent example of a rationally designed, synthetic adjuvant. Encouragingly, some promising results have already been obtained in this area, including the use of liposome- or polymerencapsulated imiquimod in combination with a TLR4 agonist [11,20]. Alternatively, the addition of fatty acid tail to imiquimod to enable lipid bilayer-based association has been described [21], as too has the chemical conjugation of a synthetic TLR7 agonist to a vaccine antigen [22]. More recently, we have described an approach in which the inherent flexibility of these small molecule adjuvants has been exploited to create small molecule immune potentiators (SMIPs) that were able to adsorb directly to Alum (Wu et al., 2014, submitted for publication). Adsorption of SMIPs to Alum allowed us to exploit all the inherent advantages of Alum, including its ability to simultaneously co-deliver antigen and immune potentiator, its low cost and wide availability as a single vial concept, and also the extensive familiarity that regulatory agencies already have with lum adjuvants. Overall, we believe that synthetic highly purified materials formulated into existing first generation adjuvants may offer the best path forward, and likely will offer significant advantages compared to the complex and heterogeneous natural products currently widely used.

7. Moving forward – developing rationally designed synthetic adjuvant formulations Since pure synthetic immune potentiators (PRR agonists) are already available, ideally they can be used to create entirely synthetic adjuvant formulations which can be included into well defined, robust, reproducible and well characterized vaccines. However, the importance of the physicochemical characteristics of the adjuvant formulation should not be underestimated. For instance, a squalene-based emulsion adjuvant induced more potent antigen specific immune responses than alternative oils [23], although squalene alone does not have an adjuvant effect [24]. Hence the characteristics of the emulsion itself are important for the adjuvant, in addition to composition. Moreover, shark derived squalene can be readily replaced with plant derived squalene [25], but the safety profile of plant derived squalene has not yet been established. Besides the oil content of emulsions, the emulsifiers used are also important, since the substitution of egg-derived phospholipids with synthetic phospholipids induced a comparable adjuvant effect, but appeared to increase emulsion stability [26]. Unfortunately, it still remains somewhat empirical to determine the best adjuvant (delivery system and immune potentiator) for each individual vaccine, and this is likely to remain so in the foreseeable future. Nevertheless, some general guidelines appear to be emerging based on accumulated experience. For example, inactivated pathogen vaccines most likely still contain inherent PRR agonists (e.g. killed or split flu vaccines and whole cell pertussis)

and may not need the addition of further TLR agonists. Likewise, emulsions or Alum alone may be sufficient for some recombinant virus-like particles (HBV and HPV). In contrast, soluble recombinant protein antigens may need a more potent and combined adjuvant, particularly if a particular kind of immune response is desired (such as a potent Th1 cell response), to protect against the pathogen. In this situation, it is clear that the adjuvant formulation may need to be optimized to obtain the preferred response, which may in some cases require antigen conjugation to the surface of liposomes [27]. Hence, our contention is that the role of formulation scientists to develop practical solutions, comprising maximum potency in alignment with acceptable stability and ease of scale up and development cannot be underestimated if we are to have greater success in future adjuvant development. 8. Does Alum have a future? Considering the extensive use of alum adjuvants, its extensive record of safety and effectiveness, and its established and inexpensive manufacturability, the obvious conclusion to this question is a clear ‘yes’, Alum will continue to be a mainstay of current vaccines. However, the real question to consider is whether or not Alum should be a key component of future vaccines? Considering the recent success of the approved alum-containing HPV vaccines and the early development work of Alum/TLR7 mentioned earlier, the obvious conclusion is once again ‘yes’, that Alum will most likely continue to be a prominent player in future vaccine development. Beyond its established safety and manufacturability, Alum’s advantages include that it is well known by the regulatory agencies, it can adsorb a wide variety of materials and can facilitate the creation of stable single vial liquid vaccines. However, while it is logical to include it in future vaccine development, there may be inherent limitations which might result in situations where emulsions and liposomes might work better. For instance, some TLR agonists and antigens may not readily adsorb to Alum, but may be more compatible with lipid-based formulations. Likewise, Alum is not suitable for intradermal delivery, if an alternative route of immunization is preferred. Moreover, Alum alone is clearly not sufficient to induce the necessary responses to protect against some challenging infectious diseases, which may require potent Th1 responses (e.g. HIV, malaria and TB). However, the addition of adsorbed PRR ligands, including TLR agonists, may overcome this limitation in some cases. It is interesting to note that in the most advanced malaria vaccine clinical trials, liposomal formulations of MPL and QS21 were preferred over emulsion-based formulations of the same components, or Alumbased formulations [28]. Perhaps future work could beneficially evaluate the effects of altering the physicochemical properties of Alum for better control of particle size and shape [29,30]. Overall, perhaps the ideal future adjuvant formulation would be completely biodegradable, with the adsorption properties of Alum, would allow thermostable single vial liquid vaccines, while stabilizing and controlling the release of vaccine components. Moreover, ideally, the adjuvant concept would also be amenable to administration by different routes of delivery, should this be desired. However, the authors are unaware of a technology that currently exists that has the potential to meet all of these desirable attributes, so the field has some way yet to go! 9. How can we improve the next generation of adjuvants? Surely some of the potential attributes that may be required for the ‘best’ adjuvant in the future may be very challenging to implement, but perhaps we do have technologies already available which can begin to be assessed to see if they can have a role in the next generation of adjuvants. For example, one

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interesting question to address is ‘can controlled release mechanisms be developed for antigens to mimic constant antigen exposure that results from virus infection and has resulted in the induction of broadly neutralizing antibodies against HIV/SIV’ (Seder et al., submitted for publication)? To induce the most effective antibodies against recombinant antigens, perhaps long term antigen exposure in vivo may be needed, also in the presence of immune potentiators. Of course, since simultaneous release of antigens and immune potentiators may be necessary, this is not a trivial undertaking, particularly considering the different physicochemical properties (including stability) of adjuvants and antigens. Nevertheless, this represents a fundamentally testable hypothesis, if existing basic science can be applied appropriately. Clearly, this work would need to be led by experienced formulation scientists working in tandem with immunologists to plan and implement these complex studies, but much long term value might emerge. Another underdeveloped aspect of vaccine formulation science concerns the potential benefits of antigen targeting, including defining which are the appropriate targets, and how best they might be accessed. Nevertheless, it should be clear that the existing first generation adjuvants induce and exploit a ‘reverse targeting’ concept as a key component of their mechanism of action [31], in which many immune cells are actively recruited to the injection site to take up the particulates. However, could this be improved by targeting specific anatomical sites or cell subsets without adding inordinate complexity and cost to the vaccine formulations? Liu et al. described an interesting approach in which vaccine antigens were targeted to the local lymph node through an albumin-based approach, but the published data involved tail vein injection in rodents, so it is not yet clear if this might work in larger species [35]. Targeted systems will require further expertise from formulation scientists, and encouragingly there is much experience and many validated concepts already available in the drug delivery field that might additionally be applied to vaccines. Another targeting-related aspect of significant interest for vaccines concerns the potential use of alternative administration routes. Specific cellular populations may be targeted more effectively through alternative routes including intradermal delivery, although tolerability may be a problem with some of the existing first generation adjuvants. Since mucosal immune responses may be necessary to protect against many enteric diseases, there remains significant interest in vaccine formulations that can be designed to have sufficient stability and bioavailability to be effective through mucosal administration. Alternatively can the addition of specific immune potentiators such as retinoic acid to standard parenteral vaccines be sufficient to cause immune cells to ‘home’ preferentially to mucosal tissues? Most likely different routes of administration may differentially shape the resulting immune responses, and perhaps combinations of routes of immunization may be necessary in some cases (e.g. IPV and OPV [32]). Once again, experienced formulation scientists will be needed to build the best systems, and perhaps to adapt existing concepts developed for other applications, including drug delivery. Even with the most skilled and effective delivery and adjuvant technologies, adjuvanted protein based vaccines will retain some limitations, and likely will need to be used in prime/boost situations with vector, or nucleic acid based vaccines for the most challenging targets [33]. Vector and nucleic acid based vaccines will likely be the ‘best’ inducers of Th1 responses and the only approach for CTL induction in man, but whether adjuvanted proteins can boost and maintain these responses merits further investigation. Some initial data are encouraging in this area (e.g. BCG boosted by adjuvanted recombinant vaccines [34,35] and viral vector boosted by recombinant protein with Alum adjuvant in the HIV Rv144 Thai trial [36]). This and other data suggests that adjuvants can help to boost the

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desired responses, even when they cannot act as an effective prime in man. 10. Analytical characterization is a key component of formulation science In this article, we have tried to emphasize the crucial role of physicochemical and biological characterization to fully understand the structure and interactions of adjuvants and antigens, for optimizing potency and stability. Much wasted time and effort unavoidably results if the design and development of novel adjuvant technologies is not accompanied and supported by a detailed analytical characterization of the formulations prepared [37]. Unfortunately, many simple and more sophisticated structural analysis tools are underutilized in vaccine development, particularly considering the complex nature of these particulate-based compositions. Thus, as a basic rule, particles should be characterized by size and morphology (e.g. dynamic light scattering, cryoTEM), component concentrations should be analyzed by spectroscopic and liquid chromatographic methods (e.g. vibrational spectroscopy, HPLC with charged aerosol detection), adjuvant impact on antigen structure should be evaluated by methods suitable for protein structure (e.g. circular dichroism, microcalorimetry, gel electrophoresis and fluorescence spectroscopy), and finally, the localization and stability of individual components should be monitored by appropriate methods. The data sets generated may have significant content, necessitating factor analysis or high powered statistical approaches. Alongside a detailed physicochemical characterization of the formulations, there is also a need for additional efforts and new tools to more fully characterize the biological mechanisms of action of adjuvants. In vitro mimic systems (e.g. VaxDesign) have demonstrated some potential, but require further development to become more informative. Tools to understand adjuvant/antigen trafficking are needed, as too are techniques to understand the influence of adjuvants on immune response broadening [38], such as massively parallel deep sequencing [39]. In many areas of physicochemical characterization and its relation to biological performance, we have barely scratched the surface so far. For example it is known that nano- and microparticles rapidly adsorb host proteins immediately following parenteral administration, meaning that immune cells encountering vaccine adjuvant particles do not ‘see’ a clean spherical surface. Instead, such particles assume a complex and evolving surface-adsorbed protein mixture immediately following immunization [40]. The relevance of this ‘protein corona’ effect for vaccine formulations remains underappreciated. While the importance of such interactions remain to be elucidated, it is clear that if formulation composition is not well controlled and reproducibly manufactured, it will be impossible to reliably characterize the interactions with the biological milieu and the resulting immune responses. 11. Conclusions Traditionally in adjuvant research there was a mix and match approach driven by biological scientists, including immunologists, in which new components were simply added to increase vaccine potency, without enough attention being paid to maximize the positive impact and minimize potential safety concerns of the added components. Unfortunately, this approach has unsurprisingly resulted in many failures. In more recent times, the key role of formulation science and analytical characterization in adjuvant development has begun to be recognized as newer second generation adjuvants have emerged (AS04, AS01, GLA-SE, Alum/TLR7, etc.). Clearly adjuvants are becoming more complex, with several

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components with very different physicochemical characteristics being included in a single formulation. Going forward, there will be a key role for formulation scientists to ensure stability, compatibility and optimal utilization of these discrete components in the final formulations, particularly in situations in which co-delivery of antigen and immune potentiators are necessary for reasons of optimal potency or safety. There are several additional attributes that we may wish the next generation of adjuvants to possess if they are to perform to even higher standards, to potentially include controlled release, immune cell targeting, or the induction of more diverse immune responses, including mucosal immunity. Formulation scientists are uniquely placed to fulfil this role and perhaps to apply advances implemented in other areas, including drug delivery, to the challenges of vaccine delivery. We hope many more formulation scientists will be prepared to join us to build the next best generation of adjuvants, since they are the people who have the key skills necessary to make it happen. Conflict of interest statement DTO is an employee of Novartis Vaccines, which has developed the MF59 adjuvant. CBF is an employee of IDRI, which has developed the GLA-SE adjuvant. References [1] European Medicines Agency. Fendrix: hepatitis B (rDNA) vaccine (adjuvanted, adsorbed). EPAR summary for the public; 2009. [2] Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, Moris P, et al. Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. J Infect Dis 2009;200:337–46. [3] Fox CB, Moutaftsi M, Vedvick TS, Coler RN, Reed SG. TLR4 ligand formulation causes distinct effects on antigen-specific cell-mediated and humoral immune responses. Vaccine 2013;31:5848–55. [4] Agger EM, Rosenkrands I, Hansen J, Brahimi K, Vandahl BS, Aagaard C, et al. Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements. PLoS ONE 2008;3:e3116. [5] Olafsdottir TA, Lingnau K, Nagy E, Jonsdottir I. IC31® , a two-component novel adjuvant mixed with a conjugate vaccine enhances protective immunity against Pneumococcal disease in neonatal mice. Scand J Immunol 2009;69:194–202. [6] Hailemichael Y, Dai Z, Jaffarzad N, Ye Y, Medina MA, Huang X-F, et al. Persistent antigen at vaccination sites induces tumor-specific CD8+ T cell sequestration, dysfunction and deletion. Nat Med 2013;19:465–72. [7] Hung IF, Zhang AJ, To KK, Chan JF, Li C, Zhu H-S, et al. Immunogenicity of intradermal trivalent influenza vaccine with topical imiquimod, a double blind randomized controlled trial. Clin Infect Dis 2014, http://dx.doi.org/10.1093/cid/ciu582. [8] Morein B, Sundquist B, Hoglund S, Dalsgaard K, Osterhaus A. Iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature 1984;308:457–60. [9] Moon JJ, Suh H, Li AV, Ockenhouse CF, Yadava A, Irvine DJ. Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction. Proc Natl Acad Sci 2012;109:1080–5. [10] Jain S, O’Hagan DT, Singh M. The long-term potential of biodegradable poly(lactideco-glycolide) microparticles as the next-generation vaccine adjuvant. Expert Rev Vaccines 2011;10:1731–42. [11] Kasturi SP, Skountzou I, Albrecht RA, Koutsonanos D, Hua T, Nakaya HI, et al. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 2011;470:543–7. [12] Singh M, Kazzaz J, Chesko J, Soenawan E, Ugozzoli M, Giuliani M, et al. Anionic microparticles are a potent delivery system for recombinant antigens from Neisseria meningitidis serotype B. J Pharm Sci 2004;93:273–82. [13] Singh M, Chesko J, Kazzaz J, Ugozzoli M, Kan E, Srivastava I, et al. Adsorption of a novel recombinant glycoprotein from HIV (Env gp120dV2 SF162) to anionic PLG microparticles retains the structural integrity of the protein, whereas encapsulation in PLG microparticles does not. Pharm Res 2004;21: 2148–52. [14] Kazzaz J, Singh M, Ugozzoli M, Chesko J, Soenawan E, O’Hagan DT. Encapsulation of the immune potentiators MPL and RC529 in PLG microparticles enhances their potency. J Control Release 2006;110:566–73.

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