Immunocorrelates of CAF family adjuvants

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Review

Immunocorrelates of CAF family adjuvants Gabriel Kristian Pedersen, Peter Andersen, Dennis Christensen

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Department of Infectious Disease Immunology, Statens Serum Institut, Artillerivej 5, DK-2300, Copenhagen S, Denmark

A R T I C LE I N FO

A B S T R A C T

Keywords: Adjuvant CAF Immunocorrelates Cationic TDB MMG

The development of the CAF family adjuvant was initiated around 20 years ago when Statens Serum Institut was preparing its first generation protein based recombinant subunit vaccine against tuberculosis for clinical testing, but realized that there were no clinically relevant adjuvants available that would support the strong CMI response needed. Since then the aim for the adjuvant research at Statens Serum Institut has been to provide adjuvants with distinct immunogenicity profiles correlating with protection for any given infectious disease. Two of the adjuvants CAF01 and CAF09 are currently being evaluated in human clinical trials. The purpose of this review is to give an overview of the immunocorrelates of those CAF adjuvants furthest in development. We further aim at giving an overview of the mechanism of action of the CAF adjuvants.

1. Development of a vaccine adjuvant for cell mediated immune responses Around 20 years ago Statens Serum Institut (SSI) was preparing its first generation protein based recombinant subunit vaccine against tuberculosis for clinical testing, but realized that there were no clinically relevant adjuvants available that would support the strong CMI response needed for effective protection against this pathogen. The Cationic Adjuvant Formulation (CAF) platform was therefore initially invented based on a need for novel safe adjuvants that could trigger a strong Th1 response [1]. The principal component of the CAF platform is the quaternary ammonium surfactant N,N-dimethyl-N,Ndioctadecylammonium (DDA) formulated into liposomes or emulsions. DDA was first demonstrated to have adjuvant properties in the mid 1960’s [2] and subsequently used the next 30 years in experimental vaccines against several viruses [3–5], bacteria [6,7] and tumors [8]. The adjuvant activity of DDA has been thoroughly reviewed by Hilgers and Snippe [9] who assessed DDA to be a moderate or strong TH2 inducer and a strong TH1 inducer. Additionally, reports from human clinical trials in the early 1970’s showed that the protection offered by tetanus toxoid adsorbed to aluminum hydroxide (Al(OH)3) is enhanced when mixed with Arquad 2 H T, in which DDA is the principal component, and that this vaccine is devoid of side effects in humans [10,11]. At SSI we had used DDA as model adjuvant since the early 1990’es where it was first used in combination with a short-term culture filtrate enriched in excreted/secreted proteins from tuberculosis and demonstrated to induce a potent cell mediated immune response of the Th1

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type [7]. Of-note it was in these studies we laid the foundation for the first TB subunit vaccine candidate Hybrid-1 (H1) by dissecting the protective role for Ag85b and ESAT-6 [6,7,12]. In 1997 Lindblad et al. showed that co-administration of IL-12 with DDA amplified the Th1 response in a dose-dependent manner and promoted a protective immune response against TB [6]. This study suggested that co-administration of IL-12 promoting immunostimulatory components would be beneficial for the Th1 induction. During the 1970’es Ribi and coworkers had developed monophosporyl lipid A (MPL) a detoxified version of LPS that retained most of the parent compound’s beneficial immunomodulatory activities [13–15], one of these being the induction of pro-inflammatory cytokines IL-1b, IL-6 and IL-12 [16,17]. In order to mimic the responses obtained by co-administration of DDA and IL-12, we thus combined DDA with MPL and showed that this significantly increased the induction of Th1 responses as compared to DDA alone [12,18]. This adjuvant, however was not stable and even short-term storage led to irreversible aggregation, making it little applicable for clinical evaluation. In a later study comparing the combination of different known adjuvants together with DDA, we found that combining DDA with the synthetic cord factor analog α,α-trehalose 6,6′-dibehenate (TDB) promotes strong protective immune responses, comparable to DDA/MPL, without overt toxicity. Shortly after we discovered, that the incorporation of glycolipids like TDB stabilizes the liposomes formed by the surfactant DDA [19] by enabling hydrogen bonding between the liposomal membrane and the surrounding water [20]. In this way, we had obtained a highly stable CMI-inducing adjuvant formulation suitable for clinical development, and CAF01 was born.

Corresponding author. E-mail address: [email protected] (D. Christensen).

https://doi.org/10.1016/j.smim.2018.10.003 Received 17 September 2018; Accepted 3 October 2018 1044-5323/ © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Kristian Pedersen, G., Seminars in Immunology, https://doi.org/10.1016/j.smim.2018.10.003

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2. Mechanism of action of CAF adjuvants

vaccine, since these responses were lost in MyD88-deficient mice. MyD88 is involved in IL-1R signaling and a partial requirement for this receptor for the Th1/Th17 responses was later found [34]. In addition, ASC mice, with deprived inflammasome activation, had reduced Th17 immunity, but maintained Th1 responses after receiving CAF01-adjuvanted vaccine [34]. Early in vitro work showed that cord factor, the natural analog to TDB was not a strong activator of human DCs [35]. Screening of the ability of apolar fractions of the Mycobacterium tuberculosis cell wall to activate human DCs resulted in the discovery of the adjuvant activity of the glycerolipid monomycolyl glycerol (MMG), suggesting that this mycobacterium derived lipid might be an even stronger adjuvant for human vaccines than TDB [35]. It was suggested that MMG also acts via MINCLE receptor [36]. Thus, replacing TDB with a synthetic analogue of MMG led to a new formulation (CAF04; Fig. 2.), which in mice gave an immune profile very similar to that of CAF01 [37,38]. The possibility of formulating additional immunostimulators to the core cationic liposomes formulation (CAF01/CAF04) is a great advantage for these adjuvant systems. Anionic molecules such as the TLR3 ligand Poly-IC [39] or lipidated agonists such as the TLR4 agonist MPL (Monophosphoryl lipid A) [40] can rather straightforward be formulated to the cationic liposomes and used to further direct and amplify the immune response. Thus incorporation of the synthetic double-stranded RNAanalogue Poly:IC (CAF05/CAF09) led to adjuvants capable of inducing strong CD8 T cell responses (see Fig. 2 and Table 1 for an overview of the composition of the different CAF’s). The specialized cross-priming CD8α(+) and CD103(+) DC subsets express high levels of TLR3 [41] and can be effectively targeted and stimulated by the cationic liposomal adjuvants (CAF05/CAF09) particularly after i.p or i.n. administration [42]. Since CAF05/CAF09 also contain the CAF01/CAF04 core (cationic liposomes with TDB/MMG, see Fig. 2) effective at inducing Th1 immunity, the magnitude of the CD8 T cell response induced by these adjuvants may be further boosted by the aid of the induced CD4 T cells [43]. In another set of experiments, it was investigated if the Th1 response induced by CAF04 could be further magnified by including additional immunostimulators. To this aim, known Th1-inducing molecules including flagellin (TLR5 agonist) and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN; TLR9 agonist) were formulated into CAF04 liposomes and tested in mice. These studies demonstrated that it was indeed possible to further boost CAF04-induced Th1/Th17 responses by including CpG ODN 1826 (CAF10), whilst flagellin (CAF11) was unable to boost these responses [44].

There are two important requirements for priming of specifically tailored immune responses: i) activation of APCs to induce a licensing signal that promotes the desired B and T cell differentiation and ii) targeting to specific APC subsets with the capacity to present antigen to the relevant cell population. It is the role of vaccine adjuvants to facilitate this activation and targeting. 2.1. Immunostimulation For the last twenty years the primary focus in vaccine adjuvant research has been directed towards the induction of licensing signals, using ligands for pattern recognition receptors (PRRs), present on APCs. Several PRRs have been defined i.e. Toll-like receptors (TLRs), NODlike receptors (NLRs) RIG-like receptors (RLRs) or C-type lectin receptors (CLRs) [21,22]. Different pathogen-associated molecular patterns (PAMPs) and synthetic analogs have been discovered to induce different types of adaptive immune responses, depending on which PRR they trigger and therefore, which cytokine environments and expression of co-stimulatory molecules are induced. Recent studies furthermore suggest that synergistic effects can be obtained by the simultaneous targeting of multiple PRRs having different downstream signaling pathways [23–30]. These combinations have e.g. been shown to increase the quality of the T- and B-cell response by inducing highavidity T- and B-cell responses, downregulating regulatory T cell responses and/or changing the antibody isotype/subset dominance. The core component of all CAF adjuvants to date is DDA, a synthetic surfactant composed of a hydrophilic cationic quaternary ammonium head group and two hydrophobic saturated C18 alkyl chains. DDA alone promotes antibody and cell mediated immune responses [9], however is physically unstable, leading to aggregation and flocculation [19] and is therefore not per se useful as adjuvant. TDB is a synthetic analog to the mycobacterial glycolipid trehalose6,6′-dimycolate (TDM). In addition to providing very effective stabilization of the DDA-based cationic liposomes, TDB acts as an immunostimulator and greatly potentiate the Th1/Th17 responses induced by DDA containing liposomes [19]. TDM is an agonist of the CLR Mincle [31,32]. Binding of TDM to Mincle leads to phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) of the FcRγ chain followed by SYK activation and Card9–Bcl10–Malt1 signalosome-dependent NF-κB signaling (Fig. 1). Thus deletion of MINCLE, Syk or any of its downstream signaling proteins abrogated responses to TDM by mouse macrophages [31]. Similarly to TDM, TDB activates the MINCLE receptor and APC activation is solely dependent on recognition of TDB by MINCLE and not by other PRRs [31,33]. However in vivo, MINCLE was not the only PRR that needed to be activated for generating antigen-specific Th1/Th17 immune responses to CAF01-adjuvanted

2.2. Vaccine delivery Newly gained knowledge on the diversity of APCs strongly suggests that in order to induce a defined immune profile by a vaccine one has to

Fig. 1. MINCLE activation by TDB/MMG-containing CAF adjuvants. 2

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Fig. 2. Overview of described CAF adjuvants and their composition.

activation processes, which is why trial-and-error have been the explorative approach of choice in the development of the vaccine delivery systems presently in clinical trials or -use. The previously mentioned new insight in the roles of innate cells, and not least the recognition of a great diversity among professional APCs dependent on their location and PRR expression pattern, opens up opportunities for rational design of vaccine delivery systems that specifically target cells which are able to facilitate the required immune response. The first generation of CAF adjuvants (e.g. CAF01-11) were designed by an empirical approach and are all based on a delivery system solely consisting of DDA liposomes stabilized with the immunostimulatory glycolipids (TDB or MMG) and in some cases additional immunostimulators. The CAF delivery system forms a strong depot at the site of injection and thus primarily targets migratory CD11c + CD11b + CD103-ve DCs [49]. This facilitates a strong bias towards MHC-II presentation and B and T-cell induction with different flavors depending on the licensing signals induced by the incorporated immunostimulators. This is, as described in detail later, also the reason why CAF09 induces a weak CD8 + T cell response after parenteral immunization, simply because the DC subset predominantly targeted by i.m immunization, has very limited cross-priming potential. Such observations led to a more goal directed design of delivery systems that could deliver the antigen and immunostimulators to locations and cell subsets of relevance. This has resulted in a number of 2nd generation adjuvants, such as CAF19 [50] and CAF24 [51], where modification of the delivery system has resulted in altered targeting profile, licensing signals and immune responses.

Table 1 CAF adjuvant nomenclature. Delivery system

CAF01 CAF04 CAF05 CAF06 CAF09 CAF10 CAF11 CAF19 CAF24

Immunostimulators

Type

Surfactant(s)

Oil

CLR-ligand

TLR-ligand

Other

Liposome Liposome Liposome Liposome Liposome Liposome Liposome Emulsion Emulsion

DDA DDA DDA DDA DDA DDA DDA DDA DDA

– – – – – – – Squalane Squalane

TDB MMG TDB TDB MMG MMG MMG MMG MMG

– – Poly-IC MPL Poly-IC CpG Flagellin – pIC

– – – – – – – – –

Abbreviations: DDA; N,N-dimethyl-N,N-dioctadecylammonium (Bromide salt). DSPC; 1,2-Distearoyl-sn-glycero-3-phosphocholine. DSPE-PEG; 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt). TDB; α,α-trehalose 6,6′-dibehenate. MMG; Synthetic monomycolyl glycerol. Poly-IC; Polyinosinic–polycytidylic acid (sodium salt). CpG; 5′-C-phosphate-G-3′ oligonucleotide. MPL; Monophosphoryl lipid A.

target APC populations with the specialty to induce that immune response [45,46]. Thus, extensive phenotyping have identified diverse APC subsets for which the PRR expression pattern interlinked with their location in the body is a determining factor for their preferential immune induction pattern [47,48]. These discoveries emphasize the importance for the delivery system to deliver the relevant immunostimulators to the right APC subset. Like for any drug delivery system the main goal for the vaccine delivery systems is to deliver the drug (here antigen and immunostimulator) to the right site within the right timeframe. In the therapeutic field, it has been a well-established practice for decades to specifically target the drugs to the organs/cells where the therapeutic effect is needed. The goal of targeted drug delivery has been to target, prolong and/or increase drug interaction with the diseased tissue. So far, a more empirical approach has been applied in the delivery of vaccines. Delivery systems like liposomes, emulsions, micro- and nanoparticles have thus been applied experimentally in vaccines for the last 50 years. A research approach based on targeted delivery has been hampered by the major gaps in the knowledge about immunological

3. The CAF series – an overview There is no rational nomenclature for the CAF adjuvant series other than chronology of invention. Thus, any numerological relations between the different adjuvants are pure coincidence. The aim for this large panel of adjuvant formulations has been to provide adjuvants with distinct immunogenicity profiles correlating with protection for any given infectious disease. The scope of this review is not to mention all CAFs, but to give an overview of the immunocorrelates of those CAF adjuvants furthest in development and for which data are already published. In the table below is thus stated only those adjuvants that will be mentioned in the 3

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4.1.2. Th17 responses A unique feature for the CAF adjuvants is the ability to induce Th17 responses. This ability was first described by Werninghaus et al. showing that CAF01 drives strong IL-17 responses by initiating signaling through the Syk-FcRγ-Card9-Bcl-10-Malt1 pathway, leading to production of proinflammatory cytokines IL-1β, IL-6, TNF-α and TGF-β [31]. Shortly after Schoenen et al. showed that this signaling happened through the interaction of the immunostimulator TDB with its cognate receptor Mincle/Clec4E [33]. It has later been shown that also MMG acts through the Mincle receptor, explaining the ability of CAF04 to induce Th17 responses [36]. Thus the CAF systems induce strong Th17 responses after parenteral immunization, which is a unique feature compared to other adjuvant systems [59]. Incorporation of other immunostimulators such as poly-IC (CAF05/CAF09) has been shown to down regulate the Th17 responses of CAF01/CAF04, whereas CpG and to some extend also flagellin (CAF11) and MPL (CAF06), has been shown to have a positive effect on the Th17 induction [44,63]. Interestingly, replacing the liposomal CAF04 formulation with a cationic squalene emulsion containing DDA and MMG (CAF19), led to a completely abolished Th17 response, although the Th1 response was similar to the liposomal counterpart. This illustrates that the delivery system has an important role for vaccines aiming to induce Th17 immunity [50]. Overall, both the CAF01 and CAF04 adjuvants have been shown to induce strong Th17 responses [33,59,63], which may be highly relevant for vaccines that should stimulate mucosal immune responses [64,65].

forthcoming sections.

4. Immunocorrelates of CAF adjuvants 4.1. CD4 + t cell induction As previously mentioned, the foundation for the CAF adjuvants was made when probing for CD4+ inducting adjuvants to support novel vaccines against Tuberculosis. The protective immunological response required against Mycobacterium tuberculosis is a field under heavy investigation. While the exact mechanism is still being elucidated, IFN-γ produced by CD4 + Th1 cells has been shown to be important for restriction of the bacterial growth [52]. In addition, Th17 cells play an important role [53,54] most probably by triggering the production of chemokines that recruit IFN-γ-producing CD4 + T cells to restrict the bacterial growth [54,55]. The hallmark for the CAF adjuvant series is therefore its ability to induce CD4 + T cell responses, especially Th1 cells and Th17 cells after parenteral vaccination.

4.1.1. Th1 responses Even though CAF01 was originally selected on its IFN-γ induction [19] it was rapidly made clear that another important immunocorrelate for the CAF adjuvants might play an important role for its ability to facilitate protection against tuberculosis, namely their ability to induce long living memory responses. High levels of protective immune responses are thus maintained for > 1 year in mice dominated by TNFα+IL-2+ and IFN-γ+TNF-α+IL-2+ multifunctional T cells with Agspecific proliferative potential and superior cytokine production profiles [56]. The ability to induce long lasting proliferative central memory Th1 cells was also associated with an increased population of KLGR1− cells, which in contrast to the KLGR-1+ cells, were found to be enriched in the lung vasculature and peripheral circulation after TB infection, suggesting that this population can readily traffic into the lung parenchyma upon exposure to pathogens and effective combat/ control infection [57,58]. A major study evaluated the immunocorrelates and of five vaccine adjuvants advanced to clinical development or already in licensed products [59]. In addition to CAF01 the adjuvants included: 1) Aluminum hydroxide used in human vaccines for more than 80 years; 2) MF59®, a squalene based emulsion used in influenza vaccines since the mid-nineties; 3) GLA-SE, similar to MF59® but also incorporating TLR-4 agonist glucopyranosyl lipid; 4) IC31®, consisting of TLR-9 agonist ODN1a anchored to cationic peptide KLK. The adjuvants were combined with M. tuberculosis; influenza and chlamydia vaccine candidates to test immune-profiles and efficacy in infection models using standardized protocols. When comparing the ability of these adjuvant systems to induce CMI responses in mice, it was found across 3 different protein antigens that CAF01 induces similar levels of CD4 T cells producing IFN-g upon re-stimulation as GLA-SE and IC31® and higher magnitude responses compared to Aluminium hydroxide and MF59® [59]. This is in line with a previous study demonstrating that CAF01 was superior to Aluminium hydroxide, montanide, MPL and CFA in inducing CD4 Th1 responses [60]. CAF04, in which TDB is replaced with a synthetic monomycolyl glycerol analog called C32MMG, which has also been shown to activate the mincle receptor [37,38], gives a very similar immunogenicity profile to CAF01, although some murine and nonhuman primate studies have suggested that it is a little more potent, and result in a more pronounced effector Th1 response [37,61,62]. Supplementation of CAF04 with TLR ligands, such as Poly-IC (TLR3) (CAF09 [62]), Flagellin (CAF11 [44]) and particularly CpG (CAF10 [44]) increased the IFN-γ secretion and % of IFN-γ secreting effector Th1 cells of CAF04 slightly, but none of the combinations altered the overall immunological CD4 T cell signature of the adjuvant.

4.2. CD8 + t cell induction Another important branch of the adaptive immune system are CD8 + T cells, capable of killing infected cells. The activation of CD8 + T cells and their subsequent differentiation into effector CTLs requires the presentation of antigen epitopes on MHC-I molecules, which usually present endogenously derived peptide epitopes. In order to obtain CD8 + T cell responses to recombinant protein antigens, these antigens have to be taken up by specialized DC subsets capable of processing antigens and presenting epitopes from exogenously derived peptides and proteins on MHC-I via cross-presentation [66]. Especially lymph node (LN)-resident CD8α+ DCs and epithelium-localized migratory CD103+ DCs play a role in cross-presentation of protein antigens to CD8 + T cells [67–70]. These cross-presenting DCs (xDCs) have a high endosomal expression of TLR3, by which signaling results in strong type I interferon responses [71]. The well known ligand for this receptor Polyinosinic–polycytidylic acid (poly-IC) and analogs like Poly-IC12U [72] and Poly-ICLC [73] have therefore been applied in numerous of preclinical and clinical trials [74]. However immunization with Poly-IC can result in unintended side effects, such as fever, malaise, hypotension, thrombocytopenia, headache etc. [74]. A reason for this is the rapid diffusion into the lymphatics of free poly-IC resulting in a systemic inflammation. In order to circumvent this and reduce systemic side effects poly-IC was incorporated into stable cationic liposomes (CAF05 and CAF09, see Fig. 2 and Table 1) [39,42,51,62,75,76]. Comparing the two adjuvants showed that CAF09 based on the combination of MMG and poly-IC was superior to CAF05 in inducing CD8 + T cell responses [62], but the immune responses induced by both adjuvants were highly dependent on the routes of administration [42,62,75]. Airway administration as well as intraperitoneal injection resulted in strong CD8 + T cell responses in addition to CD4 + T cell responses, whereas classical subcutaneous and intramuscular administration only resulted in low CD8 + T cell responses. This divergence in CD8 + T cell induction between the different administration route was demonstrated to depend on the accessibility to xDCs [42]. When vaccines containing CAF05/CAF09 is injected subcutaneously or intramuscularly a depot is formed at the site of injection. Vaccine components are then transported to the draining lymph nodes by classical CD103-ve migratory DCs and therefore do not reach lymphoid organ 4

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4.3. Induction of humoral immunity

resident cross-presenting DCs [42]. After intraperitoneal immunization on the other hand, there is a very rapid drainage of the vaccine and a major proportion ends up in draining lymph nodes and the spleen after only six hours, where they are taken up by CD8α+ve DCs as well as CD8α-ve DCs. After 24 h, the DLN is infiltrated with vaccine + migratory CD103+ DCs, most probably coming from the site of injection [42]. The peritoneal cavity thus constitutes a perfect vaccine entry site for exposure to cross-presenting DCs and thereby induction of CD8 + T cell responses against protein based vaccines [42]. This route might be acceptable for therapeutic application, such as immunotherapy in cancer treatment, but not applicable for broad vaccination campaigns. As mentioned, the depot effect is the apparent reason for the lack of CD8 + T cell responses after s.c/i.m. administration. Vaccine delivery systems based on squalene emulsions, such as MF59™ and GLA-SE has previously been shown to be delivered rapidly to the draining LN, where they are released and thus can present antigen and immunostimulators to resident DCs and macrophages [77–79]. This suggest that squalene based emulsions can be used to deliver the vaccine to LN resident CD8α+ve DCs. Poly-IC was therefore combined with the nanoemulsion adjuvant CAF19 to form CAF24. As hypothesized, the CAF24 nanoemulsions were rapidly localized in the dLNs, and the subsequent association with xDCs induced antigen-specific CD8 + T-cell responses, which were significantly higher than those stimulated by CAF09 [51]. Modifying the delivery system to target LNresident DCs instead of migratory DCs in the musculature thus facilitated a significant alteration of the induced immune response (Fig. 3).

Antibodies play an important role for protection against a number of diseases. Although the CAF adjuvants were originally developed to facilitate strong CMI responses, studies have shown that CAFs are also efficient for promoting humoral immunity against various protein antigens in mice [19,59]. CAF01 was also found to boost IgG antibodies towards influenza hemagglutinin in ferrets [80,81], HIV-1 Envelope gp140 in rabbits [82], inactivated Chlamydia trachomatis SvD bacteria and protein (MOMP) antigens in Göttingen minipigs [83] and a cocktail of Mycobacterium avium subsp. paratuberculosis in calves [84]. In humans, it was reported that the CAF01 adjuvant did not induce antibody responses significantly different from baseline to the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) [85]. The vaccine antigen and immunization/sampling regimen was however designed to promote T cell responses and not antibody responses. A recent unpublished study has demonstrated that CAF01 is at least comparable to Aluminum hydroxide in terms of eliciting antibodies against protein antigens from Malaria falciparum (GMZ2) (PACTR201503001038304, presented at DZIF annual meeting 2016, abstract T21, publication in preparation). Antibody induction by CAF01 vs aluminum hydroxide has also been evaluated in a recently finalized vaccine trial with the Chlamydia Trachomatis fusion protein CTH522 (NCT02787109, evaluation ongoing). When comparing CAF01 with other adjuvant systems for elicitation of antibody responses against three clinically relevant vaccine antigens in mice, it was found that the squalene emulsions MF59 and GLA-SE induced higher antibody responses than CAF01, particularly after the first immunization [59]. The IgG antibody titres after immunization were comparable to those observed with Alum, except the subclass distribution was shifted towards more IgG2 and less IgG1 in mice [19,59]. Thus, as opposed to other adjuvants licensed or in clinical development, CAF family adjuvants induce an antibody response skewed towards IgG2a/c, in line with the strong Th1 responses. IgG2 antibodies can bind to the FcGamma receptor 4 in mice, which has been shown to promote distinct Fc-mediated functional effects, such as antibody dependent cellular cytotoxicity, and thus CAF adjuvants open for the possibility to utilize this arm of immunity [86]. Although murine studies have shown an altered IgG subclass expression pattern after administration of antigen in CAF adjuvants, as compared to other adjuvant systems [59], it remains to be seen if this is also the case in humans. As previously described, the combination of different immunostimulators can have a positive effect on magnitude as well as quality of the induced immune response. Incorporation of multiple immunostimulators into the cationic adjuvants has indeed an effect in some cases. Poly-IC incorporation into CAF01 (CAF05) did not have a significant effect on the IgG induction [87], whereas MPL incorporation (CAF06) was found to provide a further skewing of antibody responses towards IgG2c rather than IgG1 [40]. Similar observations have been reported with squalene emulsions, where MF59™, known as an excellent adjuvant for antibody induction. Incorporation of TLR-4 ligand GLA (MPL analog) into squalene emulsions (GLA-SE) did not increase the overall IgG response but did skew towards more IgG2a [59]. Proteins/peptides formulated in CAFs can bind both to the hydrophobic membrane core or permeate through the lipid bilayer [88]. In addition, due to the highly cationic nature of CAFs, antigens may also bind via electrostatic interactions with the polar lipids. Dependent on the size and charge of the peptides or proteins in subunit vaccines, the degree of binding to the liposomes may therefore vary, which again would be expected to influence antigen retention versus drainage to lymphoid organs. Since elicitation of antibody responses requires acquisition of antigen by B cells in lymphoid follicles, the charge of the antigen could therefore theoretically affect the antibody response, although this has not been confirmed in vivo. A number of studies are currently undertaken to explore if and how antigen characteristics may influence antibody responses when matching with the different CAF

Fig. 3. A) I.p. immunization of CAF09 adjuvanted vaccine facilitates delivery of the vaccine to migratory CD103+ and lymphnode-resident CD8α+ cross-presenting DCs resulting in a strong CD8 + T cell response. Reproduced from [42] with permission from Journal of Controlled release. B) Mice were immunized three times with Ag + CAF05 either by the i.p. route or through pulmonary vaccine delivery using a Penn-Century Microsprayer aerosolizer. Non-immunized mice were included as controls. Three weeks post-immunization, pooled cells from lungs, spleen and PBMCs were isolated and non-stimulated cells subjected to ex vivo staining using PE-conjugated MHC class I-Ag dextramer and surface markers. Bars show the frequency of Ag + CD8+CD62L− cells out of CD8 + T cells in the different compartments. Reprinted from [75] with permission from European Journal of Immunology. 5

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the Th2 and Treg phenotype [92]. This limits the effectiveness of many adjuvants aiming to induce a CMI response. The PRR functions are also impaired in neonatal immune cells [93]. Thus, some PAMP-based adjuvants, e.g. many of those targeting TLRs, are ineffective in neonates despite being promising for vaccines to protect the adult population. In addition, the clinically approved non-PRR o/w adjuvant MF59 also give poor antibody and T cell responses in neonates [94]. Importantly some adjuvants seems to overcome this age related immune limitation. Notably, immunization of 1-week-old mice with a vaccine containing Ag85B-ESAT-6 in CAF01 induced IFN-g producing Th1 cells [95], thus illustrating the promise of cationic liposome-based vaccines to induce CMI responses in neonates. Neonatal mice receiving the same vaccine, given as prime (in 1-week-old mice) and boost at 4-weeks of age were protected equally well as adult mice from challenge with Mycobacterium bovis BCG i.v. In contrast, an aluminium hydroxide adjuvanted vaccine protected neither neonatal nor adult mice from the challenge [95]. The CAF01 adjuvant has also been evaluated for induction of antibody responses in neonatal mice. Whilst 1-week-old mice did not raise detectable antibody responses to a single dose of unadjuvanted influenza hemagglutinin (HA), formulation of HA in CAF01 strongly increased primary anti-HA IgG antibody responses and conferred protection against influenza challenge [96]. Notably, the antibody titres were significantly higher compared to HA formulated in IC:31 or GLA-SE [96]. As discussed above, studies comparing the same adjuvants in adult mice gave different results, as these demonstrated GLA-SE to be superior to CAF01 in terms of eliciting early antibody responses [59,96]. A deeper insight in to the elicitation of antibody responses in neonates by adjuvanted HA vaccines was obtained by measuring T follicular helper cells (TFH) and germinal center (GC) B cells. Whilst CAF01 promoted both GC B cells and TFH cells in neonatal mice, those mice vaccinated with GLA-SE and IC:31 only had TFH cells but limited GC B cell responses [96]. It is recognized that TLR activation of monocytes and DCs in newborns results in markedly different responses as compared with adult DCs, and produce Th1 polarizing cytokines and are more prone to induce a more regulatory T cell response [97,98]. A suggested mechanism behind the ability of CAF01 to induce neonatal immunity, is the CLR activation, which is also recognized as important for the Th1 induction by the BCG vaccine, which is one of the only vaccines working in neonates [99]. Thus CAF01 displays particular promise for developing vaccines to protect neonates, although in vivo data are so far still limited to mouse studies. Only CAF01 has been evaluated in neonatal studies, and it will be interesting to see how other CAF adjuvants perform in terms of inducing immunity in neonates. Dual targeting of PRRs is one strategy to increase immune responses in neonates and combining TDB with Resiquimod (R848), to enable ligation of both MINCLE and TLR7/8, led to synergistic enhancement of TNF-a production by human neonatal monocyte derived dendritic cells and increased Th1 polarization in vitro [100]. It is therefore of interest to evaluate CAF adjuvants enabling dual PRR activation in vivo as potential adjuvants for novel vaccines that effectively provide protection

adjuvants, enabling prediction of antigen suitability for eliciting optimal immune responses. Overall, it is clear that the cationic liposome formulations stabilized by TDB (CAF01) or MMG (CAF04) are efficient as antibody-inducing adjuvants. Formulating additional immunostimulators to these may potentially further increase antibody response magnitude or alter the IgG subclass profile. 4.4. Induction of mucosal IgA The ability to mount an efficient immune response at mucosal surfaces is critically important for protection against many pathogens. The current dogma is that mucosal and systemic immune systems are separated, and that lymphocyte priming in, e.g., the lungs will create strong tropism of those lymphocytes to the airways, whereas parenteral immunization induces systemic immunity. Th17 cells are recognized as an important subset for mucosal immunity and they are e.g. more abundantly present at the mucosal surface of the intestines, compared with other T-cell subsets [89]. The ability for the CAF adjuvants – especially CAF01 and CAF04 to induce strong Th17 responses after parenteral immunization is therefore interesting. In addition, especially the Th17 subset induced by CAF also expresses the mucosal homing marker CCR6 (Fig. 4C). In a recent study we found that these parenterally primed CCR6+ Th17 cells, after a mucosal recall with a homologous vaccine administered intranasal, rapidly home to the lung parenchyma and to the lung draining LNs. This rapid establishment of local mucosal Th17 responses resulted in induction of IgA and IgA producing plasma cells in the lungs (Fig. 4A and B) [64]. It was later shown in pigs that this parenteral prime- mucosal pull strategy worked both with and without the CAF01 adjuvant in the mucosal booster vaccine [90]. The primepull strategy, despite establishing local immunity in the lungs, only conferred low level secretory IgA responses in distal mucosal tissues [90]. However, it resulted in a much faster recall IgA response in the genital tract and faster clearance of C trachomatis upon intra vaginal challenge [90]. The hypothesis behind these observations is as depicted in Fig. 5, that the parenterally primed Th17 cells and co induced B cells home to the mucosal tissues in low numbers, where a mucosal boosting will facilitate a fast recall response aided by the local Ag specific lymphocytes and establish strong mucosal immunity. The prime-pull strategy using CAF01 in the priming parenteral vaccine and antigen alone in the intranasal vaccine is currently being exploited in nonhuman primates (paper in preparation) and humans as a potential Chlamydia Trachomatis vaccine (NCT02787109). 4.5. Induction of neonatal immunity Overcoming the low responsiveness of neonatal immunity poses a particular challenge for vaccine development. Many immune functions are immature or suppressed in neonates [91], and In particular the Th1 response is impaired in early life and is instead dominated by cells of

Fig. 4. Subcutaneous priming with CAF01-adjuvanted vaccine followed by mucosal boosting established lung tissue-resident IgA secreting plasma cells and CCR6 + TH17 cells: A) Agspecific IgA ELISPOT on perfused lung and spleen cells 2 weeks after booster immunization. The number of ASCs determined in triplicates from six individual mice. B) Number of IL-17+CD44+CD4+ T cells in i.n./i.n., s.c./ i.n., and s.c./s.c. immunized mice in lung parenchyma 14 days post booster immunization. C) Histogram depicting expression ofCCR6on Cyt-iv.CD45-CD44+CD4+ T cells (dashed) IL17+iv.CD45-CD44+CD4+ T cells (transparent) and IFN-γ+iv.CD45-CD44+CD4+ T cells (dark) after s.c./i.n.immunization. Reprinted from [64] Fig. 1i, 2 h and 2 g respectively, with permission from Mucosal Immunology. 6

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Fig. 5. Proposed mechanism for the induction of resident mucosal T and B cell immunity by the parenteral priming – mucosal boost strategy. The CCR6+ Th17 cells induced by the parenteral priming (left) will migrate to the lung parenchyma. After mucosal booster immunization (right) the parenterally induced parenchymal Th17 cells will engage with vaccine + mucosal APCs and rapidly be activated to induce inflammation and migrate to the local lymph node and activate T and B cell proliferation. The induced inflammation will then result in an infiltration of the induced vaccine specific lymphocytes and facilitate the establishment of a long living resident memory response in the lungs.

no. 10-092907 and 1331-00068A), The Danish Strategic Recearch Counsil (Grant no. 09-067052), Advanced Technology foundation (grant no. 007-2007-1 and 060-2009-3), Innovation Fund Denmark (grant no. 069-2011-1), ADITEC (EU grant no. 280873) TBVAC (EU grant no. LSHP-CT-2003-503367, 241745 and 643381) BIOVACSAFE (EU grant no. 115308) NIH (Grant no. AI105422 and AI124284) TRANSVAC2 (EU grant no. 730964) FASTVAC (EU grant no. 20091106) UNISEC (EU grant no 602012) FLUSECURE (EU grant no 2005207).

during early life. 5. Concluding remarks The CAF family adjuvants is at the time of writing one of few adjuvant families progressed into clinical trials with promising results, both when it comes to minimizing side effects and inducing immune responses that correlate between the different species. Besides facilitating both strong long lasting memory CMI and humoral responses, two of the important immunocorrelates for the CAF’s that makes them differ from other adjuvants are the abilities to A) induce Th17 after parenteral administration and B) induce neonatal immunity after vaccination. In comparison to most other adjuvants progressed into clinical evaluation, the CAF series adjuvants is rare because they have been developed by a public research institution and can be obtained under license for both preclinical and clinical purposes.

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Declaration of interest Gabriel Kristian Pedersen, Peter Andersen and Dennis Christensen are employed by Statens Serum Institut, which is a nonprofit government research facility, holding patents on the cationic adjuvant formulations (CAF). Acknowledgements The development of the CAF adjuvants has been supported by numerous Research grants including: the Danish Research Council (Grant 7

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