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Designing and building the next generation of improved vaccine adjuvants
Luis A. Brito, Derek T. O’Hagan
PII: S0168-3659(14)00426-XDOI: doi: 10.1016/j.jconrel.2014.06.027Reference: COREL 7261
To appear in: Journal of Controlled Release
Received date: 11 March 2014Revised date: 17 June 2014Accepted date: 18 June 2014
Please cite this article as: Luis A. Brito, Derek T. O’Hagan, Designing and building thenext generation of improved vaccine adjuvants, Journal of Controlled Release (2014), doi:10.1016/j.jconrel.2014.06.027
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Designing and building the next generation of improved vaccine adjuvants
Luis A. Brito1 and Derek T. O’Hagan1, 2
1Novartis Vaccines and Diagnostics, Cambridge, MA USA
2Corresponding author:
Derek T. O’Hagan
350 Massachusetts Ave.
Cambridge, MA 02139
Running title: Next generation of improved vaccine adjuvants
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Abstract
Vaccine adjuvants interact with the immune system, to increase the potency of vaccine antigens. Many
of the adjuvants currently available were developed with little understanding of how they worked.
Highly pure recombinant antigens are typically very poorly immunogenic due to a lack of exogenous
immune activating components such as nucleic acids, lipids, and cell membrane components. In this
review we discuss the role of adjuvants and their role as ‘delivery systems’ or ‘immune potentiators’.
We also highlight the need for appropriate delivery of immune potentiators with several ‘delivery
system’ adjuvants such as alum, emulsions, liposomes, and polymeric particles. The challenges faced by
vaccinologists to create the next generation of vaccines can be solved in-part by developing a greater
understanding of the impact of delivery, and an appreciation of the key role of pharmaceutical sciences.
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Introduction – what are vaccine adjuvants?
Vaccine adjuvants are used to enhance the potency of vaccine antigens. Although most of the adjuvants
currently available were developed empirically, without a real understanding of how they worked, this is
no longer an acceptable approach. Increasingly, modern vaccines are being developed based on
rationally designed recombinant highly purified antigens through structure based design, epitope
focusing or genomic based screening [1-4]. However, the inherent immunogenicity of these antigens is
often low in comparison to the more traditional vaccines based on live attenuated or inactivated
pathogens. Hence, there is an increasing need for potent and safe vaccine adjuvants to ensure that
modern vaccines can succeed. Moreover, there is also an increasing awareness that it is necessary to
better understand exactly how adjuvants work, how do they enhance immune responses? Nevertheless,
live attenuated or inactivated vaccines will likely continue to work effectively in the absence of
exogenously added adjuvants, since they already contain several adjuvant active components, including
non-mammalian origin nucleic acids, lipids and cell membrane components. The very practical and
pragmatic reasons why adjuvants are included in vaccines are highlighted in Table 1.
Vaccine adjuvants are defined by what they do, not what they are. The basic definition accepted by
most is that a vaccine adjuvant is something that is added to an antigen to induce a more potent
immune response. Consequently, many diverse substances have been used as adjuvants, with a very
broad range of compositions, and obtained from many sources. Nevertheless, despite the extensive
diversity of materials used, most of the established adjuvants often have more similarities than
differences. Various classifications have been proposed for adjuvants in an attempt to better understand
them, for example they are often classified into two broad groups, as ‘delivery systems’ or ‘immune
potentiators’. However recent insights into how they work, has shown that these simplistic
classifications are incorrect. In the past, we chose to define adjuvants as different ‘generations’, loosely
based on when they were first introduced and the number of components involved [5]. We believe that
this was a helpful interpretation that allowed adjuvants to be considered as more typical pharmaceutical
components of vaccines [6]. The 1st generation adjuvants are particulates of various compositions, but
similar size, including the original and the most commonly used vaccine adjuvant, insoluble aluminium
salts (alum). Most of the other established 1st generation adjuvants are formulations more familiar to
pharmaceutical scientists, since they include emulsions, liposomes and micro or nanoparticles, which
are often used for other purposes in drug delivery. For example, the first novel adjuvant to be approved
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after alum was an o/w emulsion formulation called MF59, which was followed by the introduction of a
new influenza vaccine based on liposomes [7] . Hence, the 1st generation of adjuvants were often called
‘antigen delivery systems’, since they were thought to function mainly by promoting the uptake of
antigens into immune cells. In this review we will discuss the ‘delivery’ aspects of 1st generation
adjuvants, highlighting key ‘pharmaceutical’ considerations and discuss how they might be improved.
There have been many technical innovations in drug delivery in recent years, including targeted systems
and controlled release. We will consider if some of these innovations might contribute to the next
generation of improved vaccine adjuvants? Although vaccine adjuvants will be increasingly required for
different types of vaccines, particularly for therapeutic vaccines, we will focus here on the use of
adjuvants in standard vaccines to protect against infectious disease. Other reviews in this journal will
consider the development of potential therapeutic vaccines and the technologies that might be
necessary to accomplish this significant challenge. Table 2 summarizes a subset of the adjuvants that
have been evaluated in man in clinical studies, which is only a very small subset of all the different
adjuvant technologies that have been described in the literature. Virus like particles (VLP’s) will not be
covered in this review, since they are not a vaccine adjuvant, they are ordered arrays of antigens that
are either expressed from cells or self-assemble into particles. It has been demonstrated repeatedly
that VLP’s induce stronger immune responses than soluble antigens alone, and this is one of the reasons
that commonly used vaccine adjuvants tend to be particulate structures. For example, a novel influenza
vaccine containing a HA fusion with ferritin that self-assembled into nanoparticles was recently
described [8]. The HA particles were found to induce broadly effective neutralizing antibodies to distant
influenza virus strains. However, even though the antigens were displayed in a particulate form as a VLP,
they still required the addition of an oil-in-water adjuvant. This finding is consistent with evaluations of
other purified recombinant antigens which also need added adjuvants, since they typically do not
contain the immunostimulatory molecules that are present in attenuated or inactivated vaccines. The
need for an added adjuvant is necessary for many different VLPs, and even the commercial vaccines that
contain VLPs, also contain an adjuvant, including Hepatitis B virus (HBV) and human papilloma virus
(HPV) vaccines. Interestingly, one of the commercially available HPV vaccines contains a 2nd generation
adjuvant comprising both alum + MPL, also known as AS04, from Glaxo Smith kline (GSK) [9].
Not surprisingly, the most prominent 2nd generation adjuvant approaches have tended to build
on the success of the 1st generation, since they typically comprise a first generation delivery system (e.g.
alum, emulsion, liposome or microparticle), in conjunction with one or more ‘immune potentiators’.
Tables 3a and b illustrate how potentially important delivery of both the antigen and
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immunopotentiator is for generating potent immune responses. The mammalian immune system
comprises both an innate and an adaptive component. It is the adaptive immune system with which
most people are familiar, which is responsible for long-lasting antibody and T cell responses and
immunological memory. Most current vaccines confer protection mainly through the induction of
humoral immunity, which is mediated by antibodies [10]. The adaptive immune system is the target of
vaccines and is a necessarily diverse system, since it based on an almost countless number of potential
recognition molecules (antibodies). Because of its large repertoire of recognition molecules, the
adaptive immune system is able to discriminate between and respond to an almost infinite number of
antigens. However, the successful induction of adaptive immunity not only depends on direct antigen
recognition, but also relies on essential signals that are delivered by the innate immune system [11]. The
innate immune system is our first line of host defense against pathogens and is mediated by leukocytes
including macrophages and dendritic cells (DCs) and by the complement system. It responds
immediately following infection using a limited number of germ-line-encoded pattern-recognition
receptors (PRRs) that recognize invariant pathogen-associated molecular patterns (PAMPs) that are
present on or within the organisms that infect us. PRRs function as sensors that alert the immune
system of ‘danger’, since the host (you or I) has been infected. Innate responses are typically more rapid
than adaptive responses, with responses occurring within minutes to hours, rather than days to weeks.
However, innate responses also wane rapidly, to limit the tissue damage that can result from these
potent, nonspecific effector mechanisms. Hence, innate and adaptive immunity work together in
tandem to offer maximal protection against pathogen invasion, with the ‘nonspecific’ innate system
being activated immediately on infection, while the more complex adaptive system responds more
slowly by triggering specific antibody and T cell responses. The best known PRRs , and also the ones
most commonly exploited by adjuvants, which often operate as ‘agonists’ for the PRRS, are the Toll-like
receptors (TLRs) [12]. There are at least 10 TLRs in man, which can detect infecting organisms. Typically
phagocytic cells like macrophages and DCs display TLR’s and trigger innate immunity, but they can also
function as Antigen-Presenting Cells (APCs), to trigger adaptive immunity. The most effective APC are
DCs (typically called ‘professional APC’), which digest and process antigens for presentation on the cell
surface in conjunction with MHC molecules. APCs also transport antigens to local lymphoid nodes,
where they can be presented to naive T-cells to trigger the adaptive response. Within the cytoplasm,
there are at least two additional families of PRRs: the retinoic acid-inducible gene (RIG)-like helicases
(RLHs) and the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which are also
being exploited as targets for some new generation adjuvants.
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Traditionally, vaccinologists have focused primarily on the adaptive immune response, since it
mediates protective immunity for most vaccines. In contrast, innate immunity was considered rather
less interesting until the last 15 years or so, which has seen a huge expansion in basic knowledge. This
came as a consequence of the gradual recognition that innate immunity was the key system necessary
to control and enhance adaptive immunity. The expansion of knowledge in recent years has focused on
the recognition of the different receptor systems involved in innate immunity (TLR, RIG, NOD etc), their
activation pathways and the immune mediators released. Following the initial discovery of TLRs, it
became clear that many of the established vaccine adjuvants, including the MPL included in the licensed
AS04 adjuvant were in fact agonists for PRRs. This was a huge in advance in basic science that was
recognized by the award of a Nobel Prize in medicine to one of the key scientists involved [13, 14].
However, these insights also gave rise to an unrealistic optimism that many new adjuvants would now
be easy to discover. If an adjuvant simply needs to activate innate immunity and the receptors involved
are already known, then the tools of drug discovery could be employed and high throughput screens
could be established to identify many new compounds potentially able to function as adjuvants [15].
Nevertheless, as was already established, generation 2 adjuvants tended to be generation 1 carriers in
conjunction with an ‘immune potentiator’. Perhaps the key role of the carrier was under appreciated at
this time and the contribution it would continue to make was underappreciated. Even if it was finally
clear what the immune potentiator did in 2nd generation adjuvants, which was to activate innate
immunity, there was still a key need to ‘marry’ the immune potentiator to an appropriate and effective
‘delivery system’. This review will focus on which are the delivery systems available, what are their
relative advantages and limitations, and how best can they be exploited with new generation immune
potentiators to design the next generation of adjuvants. In addition, we will consider which advances
from the field of drug delivery might be best applied to vaccine adjuvants. Can we use new concepts to
make better vaccines and which approach is most likely to succeed in the long run?
What is ‘alum’ adjuvant?
The most commonly used vaccine adjuvants in human vaccines are insoluble salts of aluminium
(alum), which have been included in many commercial products over many decades, and have been
shown to be safe and well tolerated [16]. Aluminum adjuvants are currently used in vaccines against
diphtheria, tetanus, pertussis, hepatitis B, anthrax, Hemophilus influenzae and human papilloma virus
[16, 17]. In addition, they are also used in man for immunotherapy for allergic diseases and are used in
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several commercially available animal vaccines. Although alum is the ‘archetypical’ adjuvant, it differs
significantly both structurally and compositionally from the other generation 1 adjuvants and has not
found an alternative pharmaceutical use as an injectable product. Although aluminium salts were
originally used orally as human medicines and are also used topically. The use of alum as an adjuvant is
almost a historical ‘quirk’, rather than the outcome of a considered evaluation of which material was
‘right’ for the job. Alum adjuvant was originally discovered because it was a good adsorbent on which to
purify antigens. Alum is also used orally as an adsorbent for phosphates, to prevent excessive absorption
from the intestines. Although it was not necessarily ‘built for the job’, given the established history of
safety and efficacy for alum, it is clear that it will remain a key component of vaccines in the future. In
fact, as highlighted earlier, alum has already been successfully adapted to develop a 2nd generation
adjuvant that is included in a licensed vaccine product, AS04 in HPV [18]. In marked contrast to alum,
some of the other delivery systems used as adjuvants were first used for alternative purposes, before
being adapted as adjuvants, including emulsions, liposomes and microparticles (Table 4). However,
some of the more recently described 2nd generation adjuvant concepts were designed specifically for the
purpose, e.g. Iscoms, IC31, CAF etc.
The use of aluminium based adjuvants originated in 1926 with the observations of Glenny and Pope,
who showed that alum precipitated diphtheria toxoid was more potent than the antigen alone [19]. This
observation also provided the lasting ‘definition’ of an adjuvant, something that is added to a vaccine to
enhance the immune response to an antigen. Subsequent studies also showed that alum-precipitated
diphtheria and tetanus toxoids enhanced immune responses in humans [20, 21]. However, the original
approach of ‘alum-precipitated’ vaccines has now been largely replaced for commercial products by the
preparation of pre-formed alum gels, to which the antigens can be adsorbed. This approach ensures
greater uniformity and reproducibility for the alum adjuvant. Aluminium based adjuvants are typically
but incorrectly called ‘alum’, as they will be here for brevity, but we acknowledge that this is a
simplification of the complex salts actually used. The two main types of alum used in vaccines are
typically aluminium hydroxide (AH) and aluminium phosphate (AP), which each have very different
physical and chemical compositions, and very different adsorptive behavior with different antigens.
Chemically, AH is aluminium oxyhydroxide, which has a crystalline structure and exists as small (<10nm)
needle like ‘nanoparticles’, which tend to aggregate into structures a few microns in size. The
nanometer dimensions of the primary particles result in a very large surface area for adsorption of
approximately 500 m2/g [22]. In contrast, AP is aluminium hydroxyphosphate, which has an amorphous
structure by X-ray diffraction analysis and tends to exist as ‘plates’ (50nm), which form loose aggregates
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of several microns in size. The point of zero charge (PZC) for AH is 11.4, giving the surface a positive
charge at neutral pH, while the PZC for AP varies at around 5, depending on the manufacturing
conditions, so it has a negative charge at neutral pH. In addition to the well-established and widely
available AH and AP, Merck Aluminum Adjuvant is a proprietary aluminum hydroxyphosphate sulfate
formulation (AAHS) that is structurally related to AH, as it forms an amorphous mesh-like structure and
bears a nearly zero surface charge at neutral pH [23]. In contrast to AH, AP and AAHS, Imject alum (50%
aluminum hydroxide / 50% magnesium hydroxide) has not been included in licensed vaccines, but is
commercially available for use in experimental animals. When compared with the others, Imject alum is
chemically very different, comprising crystalline magnesium hydroxide and amorphous aluminum
hydroxide, so not surprisingly, it’s performance has been shown to be significantly different. Therefore,
Imject alum should not be used in any studies that are designed to address the mechanism of action of
adjuvants [24]. Particularly since the alum actually used in a number of licensed vaccines is available for
purchase from commercial suppliers, including Brentag (http://www.brenntag-biosector.com/).
How are alum adjuvants used?
Alum adjuvants are generally used as an ‘adsorbents’ for vaccine antigens, so they generally
operate as an ‘antigen delivery system’, to promote uptake into immune cells. Although in reality, the
mechanism is much more complex. In most vaccines, antigens are adsorbed to the surface of alum
through a variety of different mechanisms to include ligand exchange, electrostatic attraction, and
hydrophobic or van der Waals forces [25]. Since proteins are complex biomolecules, often more than
one mechanism of interaction operates, and the interactions can change over time on storage, a
concept known as vaccine ‘aging’. The strongest attractive force is ligand exchange, in which phosphates
on antigens will displace hydroxyls on the AH surface [25]. So heavily phosphorylated antigens will be
strongly bound and less readily released in vivo, while antigens bound to alum by electrostatic and other
forces are more easily released. However, the degree of interaction for heavily phosphorylated antigens
can be minimized by pre-treatment of AH with phosphate buffers [25]. It is generally agreed that
antigens need to be adsorbed to alum, since low levels of adsorption has been shown to adversely
impact the immune response. However, the overall data is somewhat inconsistent, and there are
examples in which adsorption has been shown to be unnecessary [25]. Alternatively, there are examples
when too ‘tight’ a binding has been shown to negatively impact the vaccine performance [17, 26].
Nevertheless, high levels of antigen adsorption are preferred for formulation consistency, and for most
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antigens improved adsorption results in an improved immune response [26]. The need for adsorption
likely diminishes when high molecular weight antigens are used, since these are less likely to diffuse
away from the injection site. Moreover, although for many antigens stability can be improved by
adsorption to alum, there are also many examples in the literature of antigens which have been
destabilized through adsorption to alum [17, 27]. It is known that the pH of the microenvironment of the
alum surface can be different from the bulk formulation pH, due to attraction of ions, which can
contribute to protein instability. Hence defining the optimal way to use alum as an adjuvant is a multi-
factorial process that remains somewhat empirical. Extensive formulation studies are required with the
specific vaccine antigens if the maximal response is to be obtained with this adjuvant. Fortunately, we
and others have published extensively on how best to optimize the use of alum adjuvants, so extensive
information is available in the literature [6, 25, 28]. However, it is fair to say that many gaps exist in the
published information and often this area of vaccine development is considered more of an art than a
science. Nevertheless, new techniques have emerged in recent years to allow antigens to be directly
quantified on Alum and to allow quantitative and qualitative assessments in situ [21, 29-31].
Interestingly, alum adjuvant is also an effective adjuvant for DNA vaccines, but only if the DNA is
unadsorbed, AH is ineffective since the negatively charged DNA adsorbs [32].
How do alum adjuvants work?
How alum works or what is the ’mechanism of action’ is a question that has occasionally
interested, but has consistently confounded scientists for many decades since the adjuvant was first
introduced. In recent years there has been a significant expansion of work in this area, as awareness
grew of the crucial role of innate immunity in triggering and controlling the adaptive response. This
expansion in knowledge was also accompanied by the development of many new technological tools to
allow the question to be addressed in different ways. Nevertheless, although there have been one or
two apparent ‘eureka’ moments in recent years, with high profile publications resulting, in reality the
mechanism of action remains somewhat opaque. This is probably because there is no single
overwhelming contributor; the mechanism of action is complex and multifactorial, with many factors
operating simultaneously. Nevertheless, there have been several key insights in recent years which have
moved us toward a much improved understanding of how alum works. Several reviews have discussed
this work in detail, particularly from an immunological and innate signaling perspective, but full
discussion of this is beyond the scope of this review [33-35]. Here we will adopt a different approach
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and try to discuss alum from more of a pharmaceutical perspective, since a key component of the
mechanism relates to the size, shape and composition of the particles. Hopefully, our observations some
insight might contribute to the debate on how we might build a better next generation adjuvant. We will
also adopt a more pharmaceutical approach than is usually applied to compare and contrast the other
established adjuvants, and ask if they employ similar or different mechanisms of action.
Controlled or delayed release of antigen (‘Depot effect’)
Originally alum adjuvants were thought to work mainly through retention of antigen at the
injection site, allowing slow release of the antigen and an extended opportunity for interaction with the
recruited antigen presenting cells. This mechanism was proposed by Glenny et. al. and became known
as the ‘depot’ effect, but could also perhaps be more accurately termed as ‘delayed release of antigen’
[36]. The depot mechanism was initially supported by studies that showed that the tissue granuloma
induced by alum adsorbed vaccine could be removed several weeks after administration and following
maceration, be used to immunize additional animals [37]. This suggested that antigen was indeed
present at the injection site for extended time periods. However, although the depot mechanism was
accepted for many decades, it was subsequently downplayed and the importance of alternative
mechanisms was highlighted. The depot effect was first challenged by Holt, who removed the injected
antigen/alum from tissues 14 days after administration and concluded that since this had no effect on
the immune response, a long term depot was not needed [38]. Nevertheless, work conducted at the
same time showed that the use of alum adsorbed vaccines created local inflammation and ensured that
B cell blasts appeared at the injection site and the draining lymph nodes, which were present for several
weeks [39]. In contrast, more recent studies highlighted that 90% of radio-labelled antigen adsorbed to
AP was cleared from the injection site after 24 hours, although the amount of antigen that stayed local
was higher for AH adsorbed than for soluble antigen [40]. Consequently, a recent paper went so far as to
conclude that the depot is not required for alum adjuvanticity [41]. Nevertheless, as newer adjuvants
have emerged, the depot concept appears to have re-emerged, and has been assigned an important
role in the mechanism of action of CAF01 and IC31 adjuvants (discussed further below). Perhaps if we
re-define the depot effect as simply a ‘delayed release of antigen’, this allows an opportunity to
reconcile some of the apparent contradictory claims. Although long term retention of alum adsorbed
antigen at the injection site is not likely, since we know that antigen is displaced in vivo, it has been
confirmed that the alum itself is retained at the injection site, particularly the more crystalline AH [25,
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42]. In addition, other studies have also confirmed that antigen is retained at the injection site for longer
time periods if adsorbed to alum and the duration of retention is probably dependent on the
mechanisms of adsorption [25, 43, 44]. However, given the rapid cellular recruitment that results from
injection with alum, retention at the injection site for only a relatively short time frame would likely be
sufficient to ensure more extensive interaction of the antigens with APC’s. Although the recent work of
Hutchison et. al. appears to counter this suggestion, this work was done using injection into the mouse
ear, not at the usual site of vaccine administration, the muscle [41] . Moreover, if we conclude that
delayed release of antigen is attractive and a key component of the mechanism of action of alum, an
interesting question to address relates to what are the potential benefits of a slower ‘controlled release’
of antigen. Is there a preferred rate of antigen release and can it be attained in vivo?
The key role of cellular recruitment (‘reverse targeting’)
One of the key characteristic outcomes following injection of alum adsorbed vaccines is a
significant cellular infiltration into the injection site, which has been noted since the earliest studies and
was also highlighted more recently [45]. The cellular infiltration is triggered regardless of the injection
site and comprises a diverse range of immunocompetent cells to include neutrophils, eosinophils,
monocytes and dendritic cells [46-48]. In addition, older studies had shown that alum adsorption
promotes antigen uptake into monocytes and also results in activation of these cells [49-51]. The ability
of antigen rendered particulate through alum adsorption to be more effectively taken up and processed
by immune cells has been highlighted as a key role of particulate adjuvants [52]. Alum adsorption has
also been shown to increase the uptake of antigen into DC [53]. In recent studies, we confirmed in vivo
that adjuvants, including alum, are able to recruit a diverse range of immune cells into the injection site
and that these cells were able to take up antigen and deliver it to the local lymph node [48]. Hence a key
role of alum is to trigger a local inflammatory response in the muscle, which results in the recruitment of
immune cells into the injection site, which are able to process and present the antigen. The creation of a
local inflammatory response was also recently highlighted by microarray analysis and confirmed by local
cytokine production [54, 55]. We propose that this cellular infiltration could be termed ‘reverse
targeting’, since the antigen delivery system (alum and others) recruits immune cells to the site of
administration of the vaccine (figure 1). This contrasts with more established ‘targeting’ concepts in drug
delivery, in which a delivery system typically has a ligand attached which helps it to ‘find’ it’s preferred
cellular target following administration. A question that has been raised many times in the literature
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concerns the potential value of ‘targeting’ in vaccine delivery. However, we are skeptical of the potential
value of this approach. Unlike drug delivery systems which are often administered directly into the
blood stream, vaccine adjuvants are typically held within the tissue mass following IM injection, largely
based on their physical dimensions and they tend not to be ‘mobile’. This is true certainly for alum based
adjuvants, although some alternative carriers with a much smaller particle size may have greater
potential to move away from the injection site following administration. Moreover, since many immune
cells are recruited quickly to the injection site that will take up the particulates in a passive process, it is
not clear how a more ‘actively’ targeted system could offer benefits. Cellular uptake is likely controlled
by physical dimensions and surface characteristics, including size, charge, rigidity, so it is difficult to see
how much control over the uptake process could be exerted in these challenging conditions by any
targeting agent. The vaccine is present in an inflamed local tissue environment with many macrophages
and other phagocytic cells present, which have been recruited to take up the particulates. It remains to
be proven if a targeted delivery system can be any more effective than one that is passively taken up by
the many recruited cells. Moreover, it is not clear if movement away from the injection site is beneficial
for vaccine adjuvants. Perhaps smaller particles could move away from the injection site through the
lymphatics and reach the local lymph node directly, which has been claimed to be potentially
advantageous [56]. A potentially targeted vaccine delivery system could offer advantages if
administered by alternative routes, rather than the usual intramuscular route used for standard
prophylactic vaccines. For example, an innovative targeting approach from the field of cancer
diagnostics (Liu et. al.) was recently used to show that peptides adjuvanted by CpG with a lipophilic
albumin-binding domain as a targeting agent were able to effectively traffic to the lymph node and
improve immune responses after subcutaneous administration in comparison to non-targeted
formulations [57]. However, since it is the immune cells that trigger and control the immune response
in the local lymph node, perhaps it is better if antigens are delivered to the lymph node within the
recruited immune cells, which also have the ability to directly deliver the necessaryco-stimulatory
signals ? Since existing adjuvants are already very effective at recruiting immune cells to the injection
site in the muscle, which take up antigen and deliver it to the lymph node, perhaps this is already the
most efficient and effective antigen delivery approach [48]. Nevertheless, we do not exclude the
possibility that direct trafficking of an adjuvant to the local lymph node with an associated antigen could
offer potential benefits, but this needs to be demonstrated. Moreover, antigen targeting may be more
attractive when using alternative routes of administration, to include skin or subcutaneous tissue, in
which the level of cellular infiltration triggered may be significantly less than following traditional
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intramuscular injection. Hence, if we are to employ a ‘targeting’ concept for adjuvants, maybe it is
better to try to further exploit ‘reverse targeting’, in which we could promote the recruitment of a
preferred cellular population into the injection site? The recruitment factors could be co administered
with the vaccine, or could be formulation components of the vaccine which trigger the release of
recruitment factors. We have shown that a key component of the mechanism of action of the o/w
emulsion adjuvant MF59 is to trigger the release of chemokines from immune cells, which are
responsible for the recruitment of immune cells [58]. However, we are unaware of any efforts to alter
the cellular recruitment pattern triggered by a particulate adjuvant system, although this could be a
productive area of research.
Mediators that control the cellular recruitment
If we accept that cellular recruitment is a key component of the mechanism of alum, an
important question to address is what are the key mediators of this recruitment? As highlighted above,
we examined the impact of alum and other adjuvants on human immune cells in vitro and found that
they induced the secretion of chemokines (including CCL2, CCL3, CCL4 and CXCL8), which are all involved
in cell recruitment from blood into peripheral tissues [58]. Alum appeared to act mainly on macrophages
and monocytes, and triggered enhanced endocytosis and an apparent differentiation towards a
dendritic cell phenotype. These observations were consistent with other groups who also highlighted
rapid recruitment of neutrophils into the injection site, soon after alum administration [46, 55]. It was
also suggested that Mast cells can sense alum directly, and along with other cells are responsible for the
secretion of cytokines and chemokines that engender the cellular recruitment. Much earlier studies had
also highlighted that the majority of leukocytes accumulating at the injection site at early stages (<72hrs)
following alum administration were neutrophils, followed by increasing numbers of macrophages by 1
week [59]. The recruited macrophages can contribute to granuloma formation, which can persist long
after vaccination with alum [42]. Hence it is clear that alum triggers the release of various cellular
mediators following administration that result in the rapid recruitment of many diverse immune cells
into the injection site. Clearly these cells are then able to take up antigen, and to be further activated by
the presence of the alum and by other mediators released locally from the recruited cells.
Alum triggers the release of additional pro-inflammatory mediators.
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It has long been established that alum causes the release of a range of factors contributing to
local inflammation, but recent publications have outlined the mechanisms that alum uses to trigger the
release of various inflammatory substances. Early studies had suggested that alum could trigger
eosinophilia [60] and could activate complement [61]. Alum has more recently been shown to induce
the secretion of IL-1β and IL-18 by dendritic cells [62, 63]. Since this occurred in MyD88-deficient
dendritic cells, this was consistent with the observation that alum adjuvant effects in vivo were
independent of TLR signaling [64]. Alum adjuvants also cause the secretion of IL-1α by dendritic cells,
which has overlapping functions with IL-1β [65]. Of particular interest and debate in the recent past has
been the intracellular signaling mechanism by which alum triggers the release of these various
mediators. Controversy has arisen on the role and necessity of the involvement of the NLRP3
inflammasome, with consistent in-vitro data being somewhat undermined by inconsistent in vivo data.
This story has been reviewed extensively by several experts and is beyond the scope of this review [17,
33, 34, 66]. As highlighted by De Gregorio et al., perhaps the single biggest cause of the conflicting data
that has emerged is the use of non-standardized models and materials to assess the mechanism of
action of alum. The different published studies have differed significantly in their sources of alum, dose
used, routes of administration, mouse strains, antigens and regimens etc. Several recent studies have
also suggested that a variety of danger-associated molecular patterns (DAMPs) are involved in the
mechanism of action of alum, since uric acid, DNA, ATP, and HSP70 have been shown to be released by
local tissues following administration of alum [46, 67-69]. Another possible mechanism of cellular
activation by alum involves the triggering of cell surface re-assortment of lipids and the aggregation of
lipid rafts, which has been claimed to result in activation of the syk kinase and phosphoinositide-3 kinase
(PI3K) pathways [70].
A unified theory of how alum works?
As highlighted earlier, one of the most commonly asked basic research questions in vaccinology
in the last decade has been ‘how does alum work ?’ Fortunately, many new tools and techniques have
become available recently to better address this question. However, it appears increasingly unlikely that
any new ‘eureka’ insight will emerge. The mechanism of action is clearly multi factorial, with many
different components contributing collectively to the overall effect. Alum has both ‘physical’ effects due
to size, mass and composition, since it is an insoluble aggregate that triggers the influx of immune cells
that try to engulf it. But alum also has clear ‘immunological’ effects, triggering local inflammation
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through various innate signaling pathways that help to create a local ‘immune competent’ environment
in the muscle. However, it is impossible to separate the physical from the immunological effects, with
likely both operating simultaneously and acting in synergy. As already highlighted above, in addition to
immune activation by PRRs, the innate and adaptive immune systems can also be activated by
endogenous signals that originate from stressed, injured, or necrotic cells. The ‘danger theory’ of
immune activation was first postulated by Matzinger in 1994 as a key part of a new model of immunity
that suggested that immune responses are triggered by substances that cause damage, rather than by
those that are simply foreign [71]. Hence, endogenous danger signals released from damaged cells
which trigger the inflammatory response have been termed ‘alarmins’ or danger-associated molecular
patterns (DAMPs) [72]. Alarmins are thought to have three properties: they are rapidly released in
response to infection or tissue injury; they have chemotactic and activating effects on APCs, particularly
DCs; and they have potent adjuvant effects in vivo. Hence, the concept of alarmins as a danger signal
may link the physical and immunological effects of alum and better explain how these contribute to the
induction of a successful immune response. Alum acts on the tissue by generating a localized site of
inflammation, through upregulation of innate immune regulators such as cytokines and chemokines [73,
74]. Most likely alarmins may also act synergistically with microbial PRR to enhance the inflammatory
reaction following vaccine administration for 2nd generation adjuvants.
Perhaps the key lesson to learn from all the work on the mechanism of action of alum is that
many factors contribute simultaneously, including the depot effect, without any single component
dominating. This may also partially explain why so many different substances can be adjuvants, because
many different pathways can be exploited, which can converge to achieve a common effect. In addition,
perhaps we can conclude that the most successful adjuvants will likely simultaneously exploit several
alternative mechanisms, so that they can continue to work effectively in a diverse population of
individuals with a range of different antigens.
Building second generation adjuvants on alum.
Since the available vaccines are typically used in young children who are usually healthy, there is
understandably little acceptance for safety issues, or for significant adverse events. Hence the key
challenge for the development of new generation adjuvants for inclusion in vaccine products has always
been and will remain ‘safety’ [5]. Since alum is the most widely used first generation adjuvant and has
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an excellent track record of safety and tolerability, it has logically been used as a basis on which to build
second generation adjuvants. As discussed earlier, second generation adjuvants typically comprise a
delivery system (1st generation) with an added component designed to activate innate immunity (an
immune potentiator). Indeed, the first vaccine (Cervarix - offering protection against HPV infection)
containing a 2nd generation adjuvant that was approved in the US comprises a recombinant virus like
particle (VLP) antigen, which was co-adsorbed to alum alongside a TLR4 agonist, called monophosphoryl
Lipid A (MPL) [18]. More than three decades ago, the pioneering work from Ribi [75] resulted in the
development of less toxic preparations of LPS, which were subsequently further detoxified to create the
MPL, which was adsorbed to alum to create the AS04 adjuvant used in Cervarix (GSK). Studies on the
mechanism of action of AS04 have focused on the key impact of the MPL, while rather playing down the
role of the alum, which is claimed only to magnify the effect of the MPL [76]. Alum has also been
extensively evaluated in the clinic as an adsorption and co-delivery platform for other TLR agonists,
particularly the TLR9 agonist, CpG oligonucleotides [77]. However, it is important to be aware that the
adsorption of TLR agonists, particularly CpG oligonucleotides, to alum adjuvants can also negatively
impact the adsorption of antigens [78]. Hence it is necessary to co-optimize the formulation conditions
to ensure effective delivery of both the TLR agonists and the antigens in these complex combination
adjuvants, particularly if more than one antigen is present.
Overall, although it might be reasonably argued that alum was not ‘built for the purpose’ in relation to
its use in second generation adjuvants, it has many attractive attributes beyond the established safety
profile. Alum provides a solid and robust platform to which a variety of materials can be successfully
adsorbed for co delivery with antigens [25]. Since it has been demonstrated on a number of occasions
that TLR agonists require co-delivery with the antigen for optimal effect [79], it is important that alum
can be an effective delivery system for a range of components. The immune system is readily able to
induce potent adaptive responses to microbial antigens delivered to APCs in association with PRR
ligands, which is how pathogens appear to the immune system. Moreover, alum has already contributed
to the development of many stable vaccine products, so it can clearly enable the development of liquid
single vial products based on second generation adjuvants.
Several years ago, a number of small molecular weight synthetic compounds, which were
originally developed as type I IFN inducers, including imidazoquinolines (Imiquimod and Resiquimod)
and guanosine and adenosine analogs, were shown to activate TLR7 and TLR8 [80], which opened up the
possibility to discover small molecule TLR agonists. We have recently developed an encouraging second
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generation adjuvant technology in which small molecular weight immune potentiators (SMIPs) have
used novel chemistries to enable their adsorption and co delivery on alum [81]. We believe that the new
SMIP adjuvants have some potential advantages over the more ‘traditional’ large molecular weight TLR
agonists that have been used so far as adjuvants, eg. MPL. As we highlighted earlier, MPL is a detoxified
natural product with significant heterogeneity and a complex structure activity profile, which can be
difficult to formulate due to poor solubility, and shows a significant tendency to aggregate. In contrast,
SMIPs are traditional drug like small molecules which can be easily manipulated chemically to control
solubility and other solution behaviours, and are more compatible with a range of adjuvant approaches.
For example, we have modified SMIPs by the addition of a phosphonate group so that they can be
adsorbed onto the surface of alum exploiting the ligand exchange process by which many proteins
routinely adsorb [81]. The simplicity of this approach to enable alum adsorption contrasts with an
alternative formulation approach for SMIPs that was also recently described, involving chemical
conjugation of fatty acids [82]. Conjugation of fatty acids creates complex molecules that will likely
inevitably result in the preparation of heterogeneous formulations. Moreover, linking a fatty acid rather
undermines some of the most attractive features of SMIPs, since they are rendered insoluble and prone
to aggregation, like the larger molecular weight adjuvants already available. Since there are now many
different classes of SMIPs already available, and likely many more still to be discovered [15], identifying
the best approach to formulate these materials into second generation adjuvants has become a key
challenge (NIH - Blue riband Adjuvant panel report). Clearly pharmaceutical scientists are in the best
position to rise effectively to this challenge and the field of vaccinology would benefit from more
contributions from teams with diverse experience in similar drug delivery challenges.
While we believe that the discovery of SMIPs offer many advantages and are a step towards perhaps an
idealized third generation adjuvant based on molecularly defined components, an interesting question
to raise is, what should the delivery component look like ? If exploiting alum is a logical first step to
introduce SMIPs, where should we take them next? What else can we do to move towards an optimal
system, which is the best ‘delivery’, should it be a targeted system, should we control the rate of release
of the antigens. We will try to address some of these questions as we discuss the alternative approaches
to first generation ‘delivery’ based adjuvants and assess which advantages they might bring for the third
generation approach .
Emulsion adjuvants.
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Emulsion adjuvants have a long history of use in vaccine formulations, at least dating back to the studies
with ‘Freunds’ adjuvants in the 1940’s. However, although these adjuvants were very effective, they
were poorly tolerated and were not acceptable for widespread use, mainly due to the inclusion of non-
degradable mineral oils. Extensive work in the 20th century focused on a search for better tolerated
emulsion components as adjuvants. The first adjuvant emulsion comprising a fully degradable oil was
developed in the 1960s by Hilleman using peanut oil and was called adjuvant No. 65 [83]. Although this
was a step in the right direction, it took a further 30 years for the first emulsion adjuvant to be included
in a vaccine licensed for human use. MF59, which is an oil in water (o/w) emulsion comprising a low
content of the biodegradable and biocompatible oil, squalene, was first included in a human flu vaccine
in 1997 [7]. Several additional groups followed a similar path to adjuvant development and squalene is
currently the oil component of several o/w emulsion adjuvanted influenza vaccines, including Fluad®
(MF59 adjuvanted seasonal influenza), Aflunov® (MF59 adjuvanted pandemic influenza), Focetria®
(MF59 adjuvanted pandemic influenza), Prepandrix® (AS03 adjuvanted prepandemic influenza), and
Pandremix® (AS03 adjuvanted pandemic influenza). In addition to influenza vaccines, the MF59
adjuvant has also been evaluated in a number of clinical trials for a wide range of alternative vaccines,
including HSV, HIV, HBV, HCV and CMV [7, 66]. Moreover, MF59 has been extensively evaluated in a
wide range of populations of different ages, including young children, and has been shown to be safe
and well tolerated in patients as young as 6 months of age [7]. It has also been shown to allow the
development of an improved seasonal influenza vaccine in young children with significantly enhanced
vaccine efficacy [84]. Alternative emulsion adjuvants to MF59 were also approved for the first time
during the H1N1 influenza pandemic of 2009, including GSK’s AS03 and Sanofi’s AF03. However, recently
published studies have unfortunately highlighted an apparent association between narcolepsy and the
use of an A/H1N1 2009 AS03 adjuvanted influenza vaccine [85]. In contrast, despite intensive
investigations, there has been no evidence of an increased incidence of narcolepsy following the use of
any vaccines, including A/H1N1 2009 influenza vaccine, adjuvanted with MF59 [86]. It is important to
note that although the emulsion adjuvants AS03 and MF59 both contain squalene oil, they have very
different compositions, since AS03 contains the immunostimulatory adjuvant alpha-tocopherol and a
different type of influenza antigen (split virus vs. subunit) [87]. Pre-clinical studies on the mechanism of
action for AS03 showed that the presence of alpha-tocopherol induced a non-specific activation of the
immune system in the local lymph nodes of mice, in marked contrast to squalene only containing
emulsions, like MF59, which showed specific immune activation only in the muscle where the adjuvant
was injected [87, 88].
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Over the years, we have extensively investigated the mechanism of action of MF59 and have
identified key insights into how it works, which we have described in detail in a number of publications
[48, 58]. We have shown that normal tissue-resident monocytes, macrophages and dendritic cells are
activated by MF59 in the muscle and respond by inducing a mixture of cytokines and chemokines, which
results in the migration of immune cells into the injection site. The recruited cells, including monocytes
and granulocytes, produce the same factors following contact with MF59 and further amplify the
building chemokine gradient in the tissues. The outcome is a dramatically localized signal amplification
loop, which results in a significant influx of phagocytic cells into the injection site in the muscle. The high
number of cells recruited likely results in more efficient transport of antigen to the lymph nodes.
However, in contrast to alum, MF59 does not create any kind of depot and a local granuloma in the
muscle is not induced. Older studies conducted with radiolabeled MF59 showed that the adjuvant is
quickly cleared from the injection site, and that only ∼10% remains 6 h after intramuscular
administration [89]. Moreover, it was also shown that MF59 does not influence the distribution or the
half-life of the co administered vaccine antigen [90]. In addition to promoting antigen uptake by APC’s
[90], MF59 may also enhance and accelerate the differentiation of cells toward DCs and alters their
phenotype. We recently reviewed the mechanism of action of MF59 and compared it to what we know
about how alum works [88]. We noted that there are many similarities, but MF59 tends to trigger more
potent local signals in the muscle, particularly for cell recruitment, and tends to operate on different cell
populations. There are also some additional notable differences, eg. MF59 has recently been shown to
induce the release of the alarmin ATP, while alum does not [91]. We believe that ATP is a crucial
contributor to the enhanced cell recruitment and other events key to the mechanism of MF59, including
the trafficking of antigen to the local lymph node. Interestingly, we have also shown that none of the
individual components of MF59 have an adjuvant effect when used alone, it is necessary to prepare the
MF59 emulsion to have an adjuvant effect [92]. Hence, in contrast to AS03, MF59 does not have any
individual immune stimulating components, although the surfactant Span 85, does appear to contribute
to the innate signaling [92].
o/w Emulsions have also been used to develop second generation adjuvants, through the addition of an
immune potentiator. Most notably, IDRI have used a squalene based emulsion called SE (stable
emulsion), as a delivery system for a synthetic TLR4 agonist called GLA (glucopyranosyl lipid adjuvant)[93,
94]. The emulsion formulation containing GLA is called GLA-SE, its composition has varied in the
literature but primarily consists of 4% squalene (v/v), 0.72% glycerol (v/v), 0.76% phosphatydylcholine
(w/v), 0.4% GLA (w/v), and either 0.036% Pluronic F68 or 0.21% Tween 80 (w/v) in an ammonium
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phosphate buffer [95, 96]. Recent studies highlighted that SE-GLA prepared with squalene induced the
most potent Th1 responses in mice in comparison to alternative delivery technologies including, alum-
GLA, SE-GLA prepared with Miglyol, or grape seed oil, or liposomes [97]. SE-GLA has been evaluated as
an adjuvant for vaccines against a number of diseases, including leishmaniasis, malaria, influenza,
tuberculosis and HIV [93, 96-100]. While this is looking like an encouraging vaccine approach,
particularly to treat and prevent leprosy, our experiences with MF59 make us skeptical about the
suitability of emulsions as the best platform for second generation adjuvants [101]. In our hands,
although combinations of TLR agonists including TLR9 and TLR4 with MF59 looked encouraging pre
clinically [102, 103], no benefit was obtained in the clinic, at least in evaluation for flu vaccines [7].
Although adults are already seropositive to flu, so it is more difficult for additional adjuvants to have an
impact in this situation, rather than in subjects who are seronegative to the pathogen/vaccine.
Moreover, for flu vaccines it is really only the antibody response that matters, inducing a more potent T
cell response or a more focused Th1 response is not really important, although it can be for other
infectious diseases, including leprosy. Attention must be paid to the fundamental formulation science if
a robust combination adjuvant is to result, SE/GLA is prepared by dispersing, not dissolving the
components in the oil phase through sonication and heat [93] . In our hands, we have found that many
compounds, although correctly classified as lipophilic have very poor solubility in squalene (unpublished
results). Consequently, when a formulation comprising an immune potentiator in an emulsion is
prepared, the resulting emulsions can have poor stability, or heterogeneous mixtures can result, in
which the immune potentiator is not evenly distributed. Overall, although there is good evidence for
alum that co-delivery of all the vaccine components is important, the story for emulsions is less clear.
We have highlighted earlier that the antigen can be simply co-administered within the emulsion, but it
may be preferable to ensure that any immune potentiator remains associated with the oil droplets.
However, this remains an area requiring more critical evaluation on a case by case basis. An interesting
story emerged very recently that highlighted that the ‘tolerability’ (pyrogenicity) of a TLR4 agonist could
be improved by ensuring co-delivery in the oil droplet of an o/w emulsion adjuvant (AF03 – J. Haensler,
Sanofi, unpublished observations presented at IMV 2014, Albufeira). As highlighted earlier,
combinations of TLR agonists with MF59 were not well tolerated in the clinic, so we have subsequently
chosen to mostly avoid such combinations [7].
In an effort to expand the utility of emulsions for vaccine delivery, we also created and used
cationic emulsions based on squalene as a delivery system for DNA vaccines [104]and TLR9 agonist
oligonucleotides [105],which were adsorbed to the droplet surface. A similar approach was also
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exploited recently for the delivery of a new generation nucleic acid vaccine based on RNA [106]. In
addition to an adjuvant effect, the emulsion also serves to protect the RNA against degradation, and
promotes cellular uptake and sub-cellular delivery. Interestingly, the need for nucleic acids to be
adsorbed to emulsions for a positive impact contrasts sharply with the performance of alum, which was
an effective adjuvant for DNA only if the nucleic acid was unabsorbed [32].
A key characteristic for o/w emulsions in their performance as vaccine adjuvants is droplet size,
since MF59, AS03, SE and AF03 all have particle sizes between 100 and 160nm[6]. Recent advances in
particle analysis technology such as nanoparticle tracking analysis (NTA) has allowed researchers to
better understand the size distributions of mixtures [107]. The application of cryo-electron microscopy
has also allowed for direct visualization of emulsions. Since the samples are snap frozen prior to analysis
without the formation of ice, the expectation is that the morphology of the emulsion remains intact
[108]. Detailed characterization and analysis of the source of components used in emulsion preparation
also need to be monitored, since small changes can readily impact both stability and potency.
Liposomes as adjuvants.
The first report of the use of liposomes as a vaccine adjuvant was in 1974, when it was shown that
negatively charged liposomes induced an enhanced antibody response to diphtheria toxoid [109].
Liposomes were originally established as drug delivery systems and there are several products on the
market, showing that scale up and development of this approach is eminently feasible. Liposomes were
also adapted as adjuvants, but tend to be relatively lacking in potency in comparison to alternatives.
More often, liposomes have been used as a component of 2nd generation adjuvants, including AS01 [110]
and CAF01 [111]. In fact shortly after the first reported use of liposomes as vaccine adjuvants, Banerji
and Alving reported the use of liposomes for delivery of lipid A [112].
GSK has published clinical comparisons between AS01 (liposomes with QS21 and MPL) and AS02 (o/w
emulsion with QS21 and MPL) as adjuvants for malaria vaccines [113]. In initial clinical studies AS02 was
chosen as the adjuvant for the vaccine candidate after being compared against AS03 (o/w emulsion) and
AS04 (alum / MPL) [114]. However, subsequent trials comparing AS01 and AS02 illustrated that the
malaria vaccine adjuvanted with AS01 elicited stronger anti-CSP antibody responses and multifunctional
CD4+ T-cells than adjuvant AS02, which is similar to the observations in a rhesus model [113]. These
results are an excellent illustration of how delivery of immunepotentiators in different formulations can
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impact immunogenicity performance, since both formulations contained the same amount of QS21 and
MPL per dose. AS01 comprises dioleyl phosphatidylcholine (DOPC), cholesterol, and MPL, co-
formulated with QS21 in a 20 : 5 : 1 : 1 (w/w) ratio [115], whereas AS02 is composed of squalene, DL-α-
tocopherol and tween 80, which is the emulsion adjuvant AS03 [116], with the addition of QS21 and
MPL. For the malaria Phase III trial AS01 and RTS,S are simply combined as a ‘bedside’ mix [117].
Although there are no reports in the literature that we are aware of on how exactly AS01 is prepared,
patents indicate that the liposomes with MPL are prepared and added separately to a suspension of
QS21 [115]. Since it is well established that saponins such as QS21 interact with cholesterol, it would be
expected that there is an interaction between these 2 separate components in the AS01 formulation
[115, 118]. To our knowledge, similar to AS01 the details of how AS02 is prepared has not been
disclosed publicly, although the formulation appears to be a simple mixture of the preformed AS03
emulsion, with QS21 and MPL [119]. We could speculate that differences in the location in the
formulation of the immune potentiators may be responsible for the differences in potency between
these 2 adjuvants formulations (AS01 and AS02) which include the same immune potentiators at the
same dose level, although the contribution of alpha tocopherol in AS02 cannot be ignored.
Liposomes have been shown to have little to no adjuvant effect when used alone, without the
addition of an immune potentiator [120]. Although when administered at high doses IV, liposomes have
been shown to induce immune mediated hypersensitivity via the complement system [121]. Likely the
selection and optimization of the liposome composition, and dose delivered will play a role in activating
the immune system, or in avoiding activation if preferred. Liposome charge was found to impact the
ability of liposomes to induce hypersensitivity, with more negatively charged liposomes generating a
greater number of adverse events [121]. Similarly for vaccine delivery purposes, it was found that
anionic liposomes were more potent than neutral or cationic liposomes in generating Th1 responses [97].
Overall, it appears that perhaps liposomes are entering a new ‘renaissance’ in vaccine delivery, with the
field beginning to understand what impacts their potency as adjuvants, and how to use them most
effectively. In pre clinical studies, basic physicochemical parameters such as particle charge and size,
lipid membrane fluidity, membrane glass transition temperature, and the location of antigen (e.g
entrapped, surface bound, or free) have all been shown to impact potency as adjuvants [122]. If antigen
co-delivery is desired, antigens can be delivered with liposomes through a number of different
approaches, including entrapment, electrostatic binding to the surface, covalent attachment, or
membrane attachment [122-124]. Recently researchers have developed a new method to entrap
antigens and to define the location (outer membrane vs. inner membrane) of immune potentiators
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through the crosslinking of lipid bilayers that rapidly degrade in the presence of lipase [125].
Remarkably, this approach appears to induce similar levels of T-cell responses to those than can be
induced with viral vectors or nucleic acid vaccines in small animal models [125]. Further evaluation is
needed with a range of antigens and in larger species to determine if this approach can be applied
broadly, but early data are certainly encouraging. Additionally, scale up and manufacturing for this
technology remains to be established.
If in theory one can dampen the inherent signaling of liposomes by choosing the right formulation
components, does this potentially make liposomes the ‘best’ carrier for the future ? Since then we can
perhaps fully control the immune activation signal by adding the preferred activation molecules. From a
formulation design perspective this appears attractive, but in reality it may prove very difficult to
implement. Unfortunately, formulation optimization is likely to be antigen dependent, since different
antigens may interact very differently with liposomes, making it more difficult for liposomes to perform
like a broadly applicable adjuvant platform. Liposomes have been around as vaccine adjuvants for 40
years, yet their use still remains limited and so far, alum or emulsions have beeen preferred. Is this
likely to change ? Even if a generic ‘immunosilent liposome’ can be identified, which can carry immune
activation signals, questions will remain on how best to deliver many different antigens, unless it
becomes clear that antigens can be simply co-administered with liposomes and there is no need for
association. Nevertheless, liposomes like emulsions still bring the complication that it is difficult to
envisage how a single vial aqueous formulation can be defined in which all components are stable. As
discussed earlier the AS01 liposomal formulation that is in Phase III trials for malaria involves
reconstitution of the lyophilized antigen with the liposomal formulation of immune potentiators, so two
vials are necessary. While a possible solution, this is not preferred in the field, although ultimately
perhaps the liposome and the antigen can be lyophilized together, this would still require a second vial
of liquid formulation for reconstitution.
There are many examples of components that have been added to liposomes to modulate the
immune responses, including cationic lipids, TLR agonists, saponins, c-type lectin receptor agonists, and
lipids extracted from a type of single cell organisms called archaeons [122]. Each of these added lipid
immune potentiators have been shown to impact how liposomes activate the immune system. Immune
potentiators such as saponins and archaeon lipid extracts are heterogeneous mixtures, since they are
extracted from natural sources. To our knowledge archaeon lipid extracts have not been extensively
studied to determine what are the full range of active components contained. Overall, we feel that
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introducing ill-defined components into a formulation is perhaps a retrogressive step and is potentially
moving the field of adjuvants in the wrong direction. As discussed earlier, we are now entering the time
when molecularly defined adjuvants are available and we believe that these should constitute the
vaccine adjuvants of the future, rather than the ill-defined natural products of the past.
Virosomes are generally described as a liposomal adjuvant, but to our interpretation this
appears to be an inaccurate description, since they are really a different process for making a flu vaccine.
Hence the fundamental definition of an adjuvant cannot be applied, we have not added a component to
an antigen to make it more potent. In reality, following disruption of the viral membranes with
detergents, phospholipids are added to the influenza antigens and both are included in the final
product, which is a liposome. However, since the lipids are included in the final product to allow it’s
formation, it is not possible to test the vaccine antigens alone without the added adjuvant to
demonstrate an adjuvant effect. What has been created is in fact an alternative flu vaccine, with added
phospholipids replacing the natural lipids used in the viral membrane. Although the Virosomal vaccines
contain flu HA in their membranes, the same antigen is a vaccine when used alone as an inactivated
virus, and does not actually need an adjuvant to be effective in most people [126, 127]. Hence, the
description of virosomes as a liposome adjuvanted vaccine appears to be an anomaly and what we really
have is an alternative presentation of a flu vaccine which includes liposomes. Nevertheless, virosomes
are certainly as effective as alternative flu vaccines, but they appear to be lacking in potency relative to a
flu vaccine adjuvanted with an emulsion, such as MF59 [128]. Another product that uses HA virosomes is
Epaxal, a vaccine against hepatitis A virus, in which the virosomes are combined with formalin-
inactivated and purified hepatitis A virions. The HA virosomes and hepatitis A interact with one another
through electrostatic interactions. And the vaccine is stored as a liquid in a pre-filled syringe without any
additional preservatives [129]. The mechanism of improved immune responses is not clear, but there
appears to be improved T cell help and maybe innate activation from the virosome and it’s components,
to which the population has been primed due to previous exposure or immunization with flu virus [130].
Micro/nanoparticles as adjuvants
The use of PLG microparticles for vaccine delivery was first described in the early 1990’s, when
they were proposed by several groups independently as potential controlled release vaccines that could
obviate the need for booster injections [52]. However, the initial observation that polymeric
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microparticles could operate as a synthetic adjuvants was much earlier [131] and PLG microparticles
were actually first used as an oral antigen delivery systems [132]. Novel polymers had also been
proposed for controlled release of vaccine, before the more well established PLG was adopted [133].
PLG had already achieved success for drug delivery with approved products on the market [134].
However, despite extensive effort over several decades, polymeric microparticles have not yet been
successfully developed as a vaccine product, although scale up, processing and manufacturing are
already in place for this technology [135]. The potential use of PLG microparticles with encapsulated
proteins to provide sustained release was widely considered to have failed in the mid 1990’s, mainly
because the harsh process of encapsulation was not suitable for protein antigens [52]. Although, it is
conceivable to use excipients to stabilize and protect the antigen during processing, this approach has
rightly been judged a ‘mission impossible’ by most researchers, although some efforts continue [136].
Moreover, the microencapsulation approach to vaccines would be expensive relative to alternative
adjuvants, due to the need for aseptic manufacturing. Since the cost of vaccines in comparison to
therapeutic products, is usually low, the economics may not be justified for microencapsulated vaccines.
Nevertheless, a novel approach involving adsorption of antigen onto the surface of pre-formed charged
PLG microparticles was developed to overcome the limitations of microencapsulation [137]. Adsorbing
the antigen to the surface overcame the antigen instability issues, and recombinant soluble antigens
were shown to retain structural integrity, which was lost if the antigen was entrapped [138]. In addition,
immune potentiators could also be encapsulated within the PLG particles, since these are often much
more stable than protein antigens [139]. Charged PLG with adsorbed TLR9 oligo’s can also be used as a
combined adjuvant, and were shown to enhance the protective efficacy of a licensed anthrax vaccine in
an established pre-clinical challenge model [140]. An additional step forward to the potential
development of PLG for vaccine delivery involved sterilization by gamma irradiation of the pre-formed
microparticles, prior to antigen adsorption, which eliminated the need for aseptic manufacturing [141].
In contrast to Alum, PLG microparticles are comprised of biodegradable polymers which leave no tissue
residue. In addition, their safety in clinical use has been established at least for drug delivery purposes.
However, a major disadvantage of the PLG polymer is that the particles need to be lyophilized, which
adds significant expense to the product. This is in marked contrast to Alum, which typically allows the
development of stable single vial liquid formulations. In a number of pre-clinical studies in small animal
models, we have shown that PLG perhaps represents an alternative to alum for a range of established
and new generation vaccines [135]. However, although in mice PLG could trigger cross priming and
induce a CD8+ cytotoxic T cell response [142],this was not replicated in studies in non-human primates
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[143]. In addition, PLG were not more potent than the traditional alum adjuvant for the induction of
antibody responses to recombinant protein antigens in non-human primates (unpublished data). Hence,
in the long run, perhaps the biggest potential for PLG is as a co delivery system for antigen and one or
more immune potentiators, including SMIPs, which can be entrapped within the particle to which the
antigen is adsorbed [139]. Alternatively more than one TLR agonists can be co-entrapped within the PLG
with the antigen, if we agree that synergy of different TLR agonists is possible [144]. However, if this
approach is adopted, the antigen will be subjected to instability during microencapsulation and
subsequent release, as highlighted earlier [40, 135]. Moreover, this approach would require the
expensive encapsulation process and efficiencies might be low when trying to simultaneously entrap
antigens and more than one TLR agonists, with potentially very different physicochemical characteristics,
including different solubility profiles. The technical challenges for this interesting concept are quite
significant, so the advantages of encapsulation would need to be very clear and compelling. It has been
highlighted that nanoparticles have a number of potential advantages as adjuvants [56]. We and others
have extensively evaluated PLG and other nanoparticles, but the advantages in terms of potency are not
yet apparent. We described a practical process whereby nanoparticle vaccines could be created and
evaluated for vaccine delivery [145], a step that has been missing from many of the studies in this area,
but we did not see any advantages in terms of potency, at least for antibody induction, for the
nanoparticles versus the microparticles [146]. Recently an alternative way to make PLG particles that
can control the size and shape of the particles was described [147]. Using this technology rod shaped
cationic PLG particles (80nm x 320nm) were added to flu antigen [148]. Immunogenicity was
significantly improved, though no alternative adjuvants were evaluated for comparison, so it’s difficult
to get a real appreciation of the potential of the technology. We understand that this technology was
not more potent than the established flu vaccine when evaluated in a recent clinical trial (unpublished
data). Although it may be shown that the potency of PLG particles is dependent on size, we do not
believe that this has yet been clearly demonstrated. We would be excited to see high quality studies in
this area, particularly focusing on the T cell response, and perhaps using nanoparticles as delivery
systems for TLR agonists.
There are many alternative materials that have been evaluated as particulate delivery systems for
vaccine adjuvants including but not limited to polyorthoesters, polyphosphazenes, chitosan, inulin and
alginates [149-153]. The possible advantages of these systems are not immediately clear, perhaps one
can argue that a fully water soluble system could prove to be an advantage (e.g alginates), in terms of
antigen stability during formulation and antigen entrapment, but as highlighted, antigens do not need to
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be entrapped to ensure co-delivery. One aspect that has been missing from this work is a direct head to
head comparison with established adjuvants. Thus far there appears to be no clear advantages to these
systems, as other more advanced adjuvants have been able to elicit similar responses
Iscom adjuvants.
ISCOMS were originally described by Bror Morein in 1984 as a particulate carrier of Quil adjuvants and
cholesterol with membrane bound viral antigens incorporated [154], but the original technology was not
sufficiently well tolerated in man to allow commercial vaccine development. Nevertheless, extensive
modification of the technology was subsequently undertaken to mitigate the toxicity profile of Quil A,
including the concept of ISCOMATRIX (CSL), which was a pre-formed particulate with a different
composition of Quil A, to which the antigen was simply added [155]. This adjuvant concept and various
modifications with different and sometimes more highly purified Quil fractions continues to be
evaluated as an adjuvant in several settings, but mainly as therapeutic vaccines. Since the antigen is no
longer incorporated into the particulate, many more antigens can be evaluated with Iscoms, including
soluble recombinant antigens. However, the stability and perhaps the localization of the co-delivered
antigens is likely key for this adjuvant concept to be a success. The performance of Iscoms appears to be
partially dependent on antigen association, which is predominantly by electrostatic attraction forces
[117]. The newer concept of a preformed Iscomatrix with highly purified Quils at a fixed ratio was much
more attractive from a manufacturing perspective, but there may be a loss in potency when antigens
are no longer incorporated. However, newer versions of Iscoms are now available which use charged
based interactions to promote antigen association (cationic Iscoms). Mechanistically, ISCOMs appear to
destabilize endosomal membranes, which allows greater cytoplasmic access for co-delivered antigens
compared to other forms of antigen delivery [156]. Therefore, in pre-clinical studies in small animal
models, Iscoms became the gold standard adjuvant for the induction of CTL responses. They were also
able to induce CTL responses in non-human primates [157], which was a very encouraging observation,
but this could not be repeated in human clinical trials.
Rather than the emulsion, liposome and microparticle approaches discussed earlier, Iscoms are
an adjuvant technology that was really built ‘for the purpose’, they have only been used as adjuvants
and not for drug delivery purposes. However, this means that they were not able to capitalize on
process developments elsewhere in the pharmaceutical arena, any developments occurred exclusively
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in the vaccine field. GMP grade ISCOMS are prepared by solubilizing cholesterol and phospholipid in a
detergent that is subsequently mixed with saponin’s in solution, after an equilibration step, the resulting
material undergoes buffer exchange and detergent removal via dialysis or ultrafiltration; upon removal
of the detergent, the ISCOM particles spontaneously form [158]. Other preparation methods familiar
to pharmaceutical scientists such as film hydration have been evaluated, but the formulations created
have not been tested clinically [159]. Once prepared the resulting particles are stored at 4°C. This
complicates the storage of a single vial vaccine, as the antigen would also have to be stable for liquid
storage at 4°C. Since a highly purified fraction of Quil A is now included in a liposomal vaccine adjuvant
formulation (AS01) that is undergoing Phase III evaluation as a malaria vaccine, perhaps it is fair to ask
the question, if you want a Quil as a vaccine adjuvant, why not use the established liposomal approach
rather than Iscoms ? In both approaches, the Quils are bound into particulate structures through their
avid binding to cholesterol, which is a key formulation component. In both adjuvant technologies,
binding to cholesterol is a key approach to minimize the toxicity profile of Quils, a concept than has
been called Quil ‘quenching’ by GSK scientists.
Recently described adjuvant approaches – IC31 and CAF (The depot is back!).
As discussed earlier, alum based adjuvants were once thought to act mainly through the
creation of a depot at the site of injection, leading to a slow release of the antigen, but the depot effect
is in reality only part of a multi-faceted mechanism. Unlike emulsions which do not form a depot at the
site of injection [88], there have been detailed reports recently on the mechanism of action of CAF01
and IC31, two very different adjuvants that appear to require a depot effect for an adjuvant response.
Interestingly both adjuvants are able to elicit potent Th1 biased immune responses in preclinical models.
CAF01 is a cationic liposome composed of Dimethyldioctadecylammonium bromide (DDA), and
the glycolipid trehalose 6,6’-dibehenate (TDB) in a 5:1 (w/w) ratio prepared by film hydration. The
adjuvant has been tested in vaccines to protect against a wide range of disease indications including TB,
HIV, chlamydia, flu, and malaria [160-164]. The adjuvant effect of DDA has been well established since
the 1960’s, yet this molecule was never advanced as a vaccine adjuvant due to inherent instability of the
molecule [111, 165]. However, Davidsen et al. discovered that when formulated with TDB, the stability
of the DDA liposomes improved, and were more potent than DDA liposomes alone [111]. Despite the
increased liposome stability, when mixed with antigens the shelf-life of the vaccine is relatively short,
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requiring the antigen to be combined with the liposomes immediately prior to administration. Once
combined, the antigens adsorb to the surface of the liposomes to form adjuvant / antigen complexes
[166]. It has been shown that changes to the liposome composition change the biodistribution of the
adjuvant and the subsequent immune responses induced. Not surprisingly, increasing rigidity of the
liposomal adjuvant membrane by addition of cholesterol was found to impact the biodistribution of
CAF01. As cholesterol was added into the bilayer, the particles were retained at the site of injection for
an extended time, leading to an increase in the residence time of the antigen, greater activation within
the draining lymph node and an increased T-cell response [167]. Similarly PEGylation of the adjuvant
was found to inhibit the formation of a depot, negatively impacting the immune responses [168]. In fact
many changes to the composition and the biodistribution of CAF01 impacted the adjuvant response
including cationic lipid type, degree of lipid saturation, particle size and charge of the liposome, which is
consistent with what we highlighted above regarding previous liposomal adjuvants [169, 170] . To
summarize, extended muscle retention and slow release of liposome / antigen from the site of injection
favors a stronger Th1 response. However, similar to alum, we believe that this is only a part of the story,
and that this is an area that well thought out studies probing how the immune system interacts with the
liposomes could prove useful to the field.
IC31 is another adjuvant that has recently been shown to form a depot at the site of injection for
optimal immune responses. Originally designed to build on previous work on delivery of TLR9 agonists,
IC31 contains a lysine / leucine rich antimicrobial peptide KLK (KLKLLLLLKLK) derived from sapecin B, that
has a net positive charge that interacts with the negative phosphates of ODN1a [171]. The adjuvant
effect of IC31 appears to be TLR9 dependent through the induction of IFN-gamma [172]. KLK was found
to stimulate uptake and subsequent cellular internalization of ODN1a by endocytosis, similar to other
charged peptides bound with oligonucleotides [173]. Interestingly, it was shown that KLK remains on
the surface of the cell, promoting de-complexation and resulting in endosomal uptake of only the ODN,
contrary to what is observed within the field of non-viral gene therapy, where oligonucleotides are
typically shuttled into cells with the carrier [174]. Work done in-vitro with pDCs and monocyte derived
dendritic cells treated with IC31 showed a greater interaction with monocytes as opposed to MHC class
II cells [175]. IC31 is thought to work on vesicular targeting of TLR ligands by KLK and TLR9 by ODN1a in
moDCs. Spatial distribution of IC31 may be important due to increased endosomal accumulation after
repeated exposure [175]. When injected intramuscularly IC31 was found to form a depot at the site of
injection that was present for as long as 58 days after injection. [172] As one would expect, the KLK
peptide needed to be present to form the depot, and the depot was longest lived when all components
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(antigen, ODN, and KLK) were present [171, 172]. It is unclear what the advantage is to retaining an
antigen at the site of injection for 58 days, when there are other adjuvants that allow for faster
clearance. Additionally, is an extended depot necessary for the adjuvant response of IC31, as it has
been shown that activation of a soluble TLR9 agonist in the presence of a soluble antigen leads to
improved immune responses, though co-delivery of antigen and adjuvant appears to further improve
responses [176]. Some well-designed experiments to determine how critical the depot effect is for the
subsequent immune response would be highly beneficial to the field.
How do we design the next best generation of adjuvants ?
In this section we would like to take the opportunity to highlight a number of questions that we
believe are important and worthy of further consideration, if we are to get closer to defining the next
best adjuvant technology. By asking the questions, we obviously do not believe that an answer is yet
available, but we will attempt to offer our own views of the most likely answers based on the currently
available knowledge. The main purpose for raising these questions is to highlight areas that might be
benefit from some well-designed and appropriately controlled basic research studies.
Do we need targeted adjuvant systems, would they perform better than those that we currently
have? If we believe that the answer is yes, which are the right targets and where do they reside, which
are the receptor systems that can be exploited? As discussed earlier, we are skeptical of the value of
targeting for adjuvants which will be injected into the muscle, since they already benefit significantly
from ‘reverse targeting’, the adjuvant triggered influx of immune cells. So it’s difficult to imagine how a
targeted system could offer benefits when most of the particles are taken up anyway. However, in
theory we could promote particle interaction with a specific cell population that is preferentially
recruited to the injection site and this could potentially bring benefits. Moreover, this is a concept that
can be tested in pre-clinical studies, since targeting agents for different sub populations of immune cells
are readily available. Nevertheless, perhaps a targeted adjuvant system would likely be more beneficial
if delivered into an alternative site, such as the skin, which has many immune competent cells already
present, unlike the muscle where they need to be recruited? Again, this is a testable hypothesis worthy
of investigation in pre-clinical models.
Another good question to address is whether or not there is a new generation of ‘carrier’ that
can replace what is currently available? Many adjuvants have been around for quite some time and have
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been successfully scaled up and developed commercially (Alum, emulsions and liposomes), while some
newer adjuvants have only emerged in recent years (CAF01, IC31 etc.), and are supposedly ‘purpose
built’. Are the newer ones more potent than the ones we had already? We think this remains an open
question, but one that can only be addressed with carefully controlled human studies; fortunately such
studies are already underway. Nevertheless, it should be clear that for adjuvants, potency is certainly
not the only question, safety is actually far more important [5]. In addition, there are many other key
questions to address for the new approaches beyond safety and tolerability, including their ability to be
successfully scaled up, cost of goods, stability with different antigens, suitability for a wide range of
antigens etc. If we are clear that we want to accomplish co-delivery of antigen and immune potentiator
to the same cells, is a formulation approach preferred, or could this be better accomplished by
chemically conjugating the immune potentiator to the antigen [177]. What are the respective pro and
cons of the conjugation approach versus the more established formulation based approaches? As our
understanding of the immune system improves, along with a broader appreciation of its key triggers and
controls, the potential to harness it more effectively, increases not only for preventing infectious
diseases, but possibly also for ‘therapy’. However, it is clear that there will be a need for much more
potent adjuvants to enable the development of therapeutic vaccines, particularly if it is necessary to
overcome ‘tolerance’ to enable cancer vaccines. In addition, the acceptable tolerability and safety
profile of adjuvants will be very different for therapeutic vaccines to be used for people already sick,
rather than standard prophylactic vaccines, which are administered to healthy people. Therefore, a
much broader range of adjuvant technologies than those highlighted here could have a key role in
therapeutic vaccines, particularly if a range of immune potentiators is included. Nevertheless, even the
most potent adjuvant technologies may not be effective, without combination with alternative
approaches such as live vectors or nucleic acids, which will be much more potent for the induction of
CD8+ responses, which likely will be necessary. Hence in many areas, the potential value of new
adjuvants in a prime/boost setting needs to be addressed, in which a potent immune response is already
triggered by a viral vector or a nucleic acid. It is not yet clear which are the best adjuvants to use in this
setting, or if the most potent adjuvants to prime a response in an antigen naive animal will still be the
best to boost that response when the animal, or human, is already ‘heavily’ primed by a vector or
nucleic acid ?
Additional questions worthy of consideration for which we believe answers are not yet available
include the following; which is the best available delivery system to accomplish co delivery of antigen
and TLR agonists? Should we combine TLR agonists, which ones, or should they be combined with
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alternative compounds that activate different elements of innate immunity? Do we need controlled
release, for the antigen, or for the TLR agonist? One key lesson from studies of live attenuated vaccines
is that activation of multiple innate receptors simultaneously is more effective than activation of a single
pathway [178]. Therefore, an optimal vaccine candidate may require the delivery of several TLR agonists
within the same delivery system, in conjunction with the antigens. Alternatively, we may need to deliver
selected TLR agonists in conjunction with additional agonists which activate different pathways of innate
immunity. If we want controlled release, do the antigen and the TLR agonist need to be released at the
same time, or differently? Given the likely different physicochemical characteristics of antigens and TLR
agonists, is there a delivery system with enough flexibility to accommodate co delivery of both agents
simultaneously? Do we know which controlled release profile is preferred, for antigen and TLR agonists,
and how do we get it? Will any preferred release profile that is identified be antigen and adjuvant
dependent, and not universally applicable? If vaccines need mucosal immunity do we have to design an
effective mucosal delivery system with all the associated challenges, or can we induce mucosal
immunity by adding excipients such as retinoic acid to a standard injectable formulation [179]?
Alternatively could metabolites of vitamin B be the most effective way to induce mucosal immunity, but
how should we administer them [180]?
We believe that many of these questions will be best addressed by basic researchers probably
based in academia or the public sector, but this work will need to be collaborative. Formulation
scientists can potentially build a broad range of delivery systems, comprising targeted systems and those
with controlled release characteristics, but they need to collaborate extensively with immunologists and
other scientists to determine what works best. Unfortunately, vaccinology remains an inexact science,
with many basic concepts still not well understood. Even ‘immunogenicity’ is a poorly defined
phenomena, since we often have a limited understanding of why certain antigenic components are
‘immune dominant’, while others are more silent? However, we would contend that pharmaceutical
scientists have a key and distinct role to play in designing the next generation of adjuvants. We hope
that with this discourse on the many ‘unknowns’ we have helped to begin to define a framework of
what is currently known, which is often not nearly enough, and we have encouraged more basic
researchers to help to seek solutions to these complex problems.
Conclusions
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For too long vaccine adjuvants have been a somewhat ill-defined and poorly understood area in
science, particularly mechanistically, but we are at a point in vaccine development when they need to
become as well defined as other pharmaceutical formulations. Although there have been significant
improvements in the relatively recent past, with new fully characterized adjuvant formulations included
in licensed vaccines, there are still too many examples of poorly characterized adjuvants, some of which
are still progressing into clinical evaluation. Even if these adjuvants perform well in the clinic, they risk
repeating the failures of the past and not progressing beyond initial evaluations. We believe that
vaccine adjuvants need to be considered like any other pharmaceutical formulation, and the
fundamental considerations should be unchanged. If they are to succeed and to be eventually included
in licensed products, adjuvants need to be robust, scaleable and reproducible. In addition, they need to
display these characteristics in conjunction with the vaccine antigens with which they will be co-
formulated, to create a vaccine product. Hence it should be clear that there is a key role to play for
pharmaceutical scientists in contributing to the next generation of rationally designed adjuvants. In our
opinion, the role of pharmaceutical scientists in the discovery and development of vaccine adjuvants has
been traditionally under-appreciated, which may partially explain the many failures in this area. The
identification of adjuvant active compounds has been over emphasized, while the need to create an
appropriate formulation with the compounds has been less well understood. We would like to take this
opportunity to encourage all vaccinologists to reach out to pharmaceutical scientists to help to solve the
complex formulation problems of vaccine adjuvants. In addition, we would like to encourage our fellow
pharmaceutical scientists to be more receptive to the challenges and demands of vaccine adjuvant
discovery and development. We believe that pharmaceutical scientists are uniquely positioned to solve
some fundamental problems in this area and can become a key part of the vaccine research and
development enterprise. The authors believe that formulation science is still under represented in
vaccinology, and there are many areas in which we could help. Pharmaceutical scientists could use
existing expertise to lead efforts in screening for new small molecules as adjuvants, or modify the
compounds appropriately to ensure that they have the right features for retention within the
formulation and effective co-delivery with the antigens. They could also lead efforts to design the next
delivery system for antigens and immune potentiators, or work on ensuring antigen stability and optimal
immunogenicity, or even facilitate vaccination by a preferred needle free route of administration. They
could also use existing expertise to determine which is the optimal release profile for immune
potentiators, or to target them to a preferred population of cells, to determine if this is indeed
advantageous. Since many of the challenges in vaccine delivery/formulation are similar to those for a
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variety of ‘drug delivery’ problems, particularly therapeutic proteins, there is already an enormous
wealth of experience waiting to be applied to solve the problems of vaccine delivery. We would like to
take this opportunity to encourage our colleagues both in the pharmaceutical sciences and in vaccines
R&D to engage more fully with one another, to use their respective experiences to help to design the
next generation of vaccines.
Although at this point, it is not yet clear which is the ‘best’ approach in the long term for vaccine
delivery, we would suggest that if we try to use the most established systems (liposomes, emulsions,
microparticles etc), we will get access to all the advances that have already been made for these
technologies for drug delivery. In addition, we will also get access to any future improvements, which is
an area of considerable investment in basic and applied science. Importantly, by focusing on these
established technologies, we potentially also get access to many of the talented minds who have already
worked on the key challenges, including scale up, manufacturing and the development of aseptic
processes etc. The recent success of AS04 adjuvant, including inclusion in a licensed vaccine in the US, is
perhaps a very good example of the advantages of ‘building on success’. This approach utilized a well-
established vaccine adjuvant platform (alum) and adsorbed the TLR4 agonist, to ensure that the
development risks were lowered. We believe that a similar approach can also be applied for other TLR
agonists and will bring many benefits to vaccine development. However, we also believe that there are a
number of alternative drug delivery systems which potentially have inherent advantages over alum and
may be adapted for vaccine delivery. We look forward to hopefully welcoming the endeavor of many
more skilled pharmaceutical scientists to this undertaking in the future.
Figure and table legends:
Tables 3a and b. Synergy between an particulate antigen delivery systems and immune potentiators
(TLR agonists)
We used a soluble recombinant antigen from Neisseria meningitides serotype B (Men B) to illustrate the
value of rationally combining different adjuvants to ensure effective co-delivery of an immune
potentiator (TLR agonist) with the vaccine antigen. In Table 3a, we show in a mouse study that the
soluble recombinant antigen is a typically poor immunogen alone, which was not significantly improved
either by the addition of a TLR9 agonist (CpG) or by conjugation to a protein based TLR5 agonist
(bacterial Flagellae). All of these approaches induced low level of binding antibodies and did not elicit
functional antibodies which can kill the bacteria, bactericidal antibodies. However, if the antigen is
adsorbed to a PLG microparticle, it becomes an effective immunogen, which can induce bactericidal
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antibodies, which are the correlate of protective immunity against this pathogen. Moreover, the titer
can be significantly enhanced if an immune potentiator (CpG) is then added to the formulation. In Table
3b, we take the co-delivery concept to the next level, showing combined delivery of the antigen and a
different TLR agonist, MPL (TLR4) with PLG. Like CpG, MPL can be added to the PLG microparticle with
adsorbed antigen to enhance the level of response, but the response is further enhanced if the PLG
microparticle also simultaneously delivers the antigen and the TLR agonist. In this situation, this was
best accomplished by adsorbing the antigen to the PLG, which already had entrapped MPL. Similar data
has been obtained with alternative delivery systems, including the better established Alum.
Figure 1:
Schematic representations of the concept of ‘reverse targeting’, a key concept in the mechanism of
action of MF59 o/w emulsion adjuvant. Antigen and MF59 emulsion adjuvant are administered into
muscle that does not contain resident immune cells (a). Adjuvant creates local immune competent
environment by interacting with cells to trigger release of recruitment factors (e.g. chemokines) (b).
Recruitment factors attract immune cells to the site of injection, immune cells take up antigen (c).
Adjuvant acts on recruited cells to enhance cellular recruitment (d). Additional cells infiltrate muscle,
take up additional antigen, and traffic to lymph node (e).
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Table 1.
Why adjuvants?
Increased antibody titers
Induce responses more rapidly
Allow antigen dose reduction or fewer doses
Increased breadth of response to overcome pathogen diversity
Induce long-lasting immune memory responses
Overcome poor immune responses in elderly and young children
Table. 2. Which adjuvants do we have ?
Adjuvant Description Clinical
status
References
Aluminum salts Insoluble aluminum salts eg.
phosphates and hydroxides. Have an
extensive safety record and are being
used as a platform for second
generation adjuvants. Antigens are
typically adsorbed to the surface.
Licensed in
US and EU
[16]
MF59 Squalene oil in water emulsion
adjuvant that has been part of a
licensed flu vaccine since 1997.
Antigens are not associated with
emulsion droplets. extensive safety
record.
Licensed in
EU
[88]
AS03 Squalene oil in water emulsion
adjuvant with the added immune
potentiator alpha tocopherol, used in
flu vaccines during 2009 pandemic,
but associated with narcolepsy.
Licensed in
EU and US
[85] [87]
AS04 Combination of aluminum adjuvant Licensed in [181].
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with the TLR 4 agonist
monophosphoryl Lipid A (MPL) co-
adsorbed. Approved as a licensed HPV
vaccine (Cervarix)
EU and US
Virosomes Influenza virus envelopes
reconstituted in phosphatidylcholine
bilayers (virosomes).
Licensed in
EU
[134]
AS01 Liposomes composed of dioleyl
phosphatidylcholine (DOPC),
cholesterol, MPL and QS21 in a 20 : 5 :
1 : 1 (w/w) ratio.
Phase III
Malaria
[115]
CAF01 Cationic liposome composed of
Dimethyldioctadecylammonium
bromide (DDA), and the glycolipid
trehalose 6,6’-dibehenate (TDB) in a 5:1
(w/w) ratio prepared by film hydration.
Phase I
[182]
Poly I:C Synthetic double stranded
oligonucleotide containing repeating
units of inosine and cytosine that
signals through TLR3.
Phase I [183]
IC31 Antimicrobial peptide KLK bound to
ODN1a, signals through TLR9.
Phase I [171]
AS02 Co-mixture of AS03 emulsion adjuvant
with QS21 and MPL.
Phase II [116, 119]
Imiquimod Small molecules immune potentiators
that signal through TLR7 / 8.
Phase II [184]
CpG
oligonucleotides
(ISS 1018)
TLR9 agonists based on bacterial DNA. Phase III [176]
SE / SE-GLA Squalene based emulsion that is a
stand-alone emulsion adjuvant, or
combined with the TLR4 agonist GLA.
Phase I [155, 185]
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ISCOMS and
ISCOMATRIX
Small (40nm) lipid based adjuvants
consisting of phospholipids,
cholesterol and saponins.
Phase I [186]
Table 3a
Groups Serum IgG Bactericidal titer
Soluble Men B antigen 423 <16
Men B / TLR5 fusion 11 <16 Men B + CpG 511 <16
PLG adsorbed Men B 23,171 64 PLG adsorbed Men B + CpG 137,747 1,024
Table 3b
Groups Serum IgG Bactericidal titer
PLG adsorbed Men B 11,367 512 PLG adsorbed Men B + MPL 18,074 2048 PLG entrapped MPL and adsorbed Men B 66,493 8192
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Table 4.
Delivery
system
First
described in
literature
First medical
uses
First approved for
use as drug delivery
vehicle
First approved
for use as
adjuvant in
vaccine
Emulsions Early 1900’s
[187, 188]
Intralipid –
1962 [189]
Propofol – 1989 [190] MF59 – 1996 [7]
Liposomes 1964 [191] Doxil – 1995
[190]
Doxil – 1995
[190]
Invlexal V –1997
[192]
Microparticles
(PLGA)
Early 1970’s
[134]
Decapeptyl
LP – 1986
[134]
Decapeptyl LP – 1986
[134]
N/A
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Figure 1
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Graphical abstract
